US6090392A - HIV envelope polypeptides and vaccine - Google Patents

HIV envelope polypeptides and vaccine Download PDF

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US6090392A
US6090392A US08/889,841 US88984197A US6090392A US 6090392 A US6090392 A US 6090392A US 88984197 A US88984197 A US 88984197A US 6090392 A US6090392 A US 6090392A
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Phillip W. Berman
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Genentech Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • This invention relates to HIV envelope polypeptides and vaccines containing the polypeptides.
  • AIDS Acquired immunodeficiency syndrome
  • HIV human immunodeficiency virus
  • Subunit vaccines generally include gp120, the portion of the HIV envelope protein which is on the surface of the virus.
  • the HIV envelope protein has been extensively described, and the amino acid and nucleic acid sequences encoding HIV envelope from a number of HIV strains are known (Myers, G. et al., 1992. Human Retroviruses and AIDS. A compilation and analysis of nucleic acid and amino acid sequences. Los Alamos National Laboratory, Los Alamos, N. Mex.).
  • the HIV envelope protein is a glycoprotein of about 160 kd (gp160) which is anchored in the membrane bilayer at its carboxyl terminal region.
  • the N-terminal segment, gp120 protrudes into the aqueous environment surrounding the virion and the C-terminal segment, gp41, spans the membrane.
  • gp160 Via a host-cell mediated process, gp160 is cleaved to form gp120 and the integral membrane protein gp41. As there is no covalent attachment between gp120 and gp41, free gp120 is sometimes released from the surface of virions and infected cells.
  • the gp120 molecule consists of a polypeptide core of 60,000 daltons which is extensively modified by N-linked glycosylation to increase the apparent molecular weight of the molecule to 120,000 daltons.
  • the amino acid sequence of gp120 contains five relatively conserved domains interspersed with five hypervariable domains. The positions of the 18 cysteine residues in the gp120 primary sequence, and the positions of 13 of the approximately 24 N-linked glycosylation sites in the gp120 sequence are common to all gp120 sequences.
  • the hypervariable domains contain extensive amino acid substitutions, insertions and deletions. Sequence variations in these domains result in up to 30% overall sequence variability between gp120 molecules from the various viral isolates. Despite this variation, all gp120 sequences preserve the ability of the virus to bind to the viral receptor CD4 and to interact with gp41 to induce fusion of the viral and host cell membranes.
  • gp120 has been the object of intensive investigation as a vaccine candidate for subunit vaccines, as the viral protein which is most likely to be accessible to immune attack. At present, clinical trials using gp120 MN strain are underway. However, to date no human vaccine trial has been of sufficient size to confirm or refute vaccine efficacy.
  • candidate HIV-1 vaccines are burdened by the lack of in vivo or in vitro models of HIV-1 infection that accurately approximate the conditions of natural infection in humans.
  • candidate HIV-1 vaccines [Berman et al.; J. Virol. 7:4464-9 (1992); Haigwood et al.; J. Virol. 66:172-82 (1992); Salmon-Ceron et al.; AIDS Res. and Human Retroviruses 11:1479-86 (1995)] have been described that elicit broadly cross-reactive antibodies able to neutralize a variety of diverse HIV-1 isolates in vitro.
  • the relevance of in vitro assays to protective immunity in vivo is uncertain.
  • HIV-1 infection in vivo and in vitro both suffer from the limitation that the in vitro amplification of HIV-1, which is required to prepare virus stocks for in vitro or in vivo infectivity experiments, imposes a genetic selection that results in a spectrum of virus quasi-species that differ from the spectrum of variants present in the clinical specimens used to establish the culture [Kusumi et al.; J. Virol. 66:875 (1992); Meyerhans et al.; Cell 58:901-10 (1989)].
  • One of the candidate HIV-1 vaccines that have entered human clinical trials is recombinant gp120 prepared in Chinese hamster ovary (CHO) cells from the MN strain of HIV-1 (MN-rgp120) (Berman et al.; J. Virol. 7:4464-9 (1992)). To date, approximately 499 adults have participated in Phase 1 and 2 immunogenicity and safety trials of this vaccine.
  • MN-rgp120 is safe, immunogenic, and elicits high titers of neutralizing antibodies in greater than 95% of individuals immunized according to a 0, 1, and 6 month immunization schedule [Belshe et al.; JAMA 272(6):475-80 (1994); McElrath; Seminars in Cancer Biol. 6:1-11 (1995)].
  • nine vaccinees who received MN-rgp120 have become infected with HIV-1 through high risk behavior.
  • Small trials, such as these, in populations with low rates of infection and minimally sized placebo control groups do not have sufficient statistical power to confirm or refute vaccine efficacy.
  • Recombinant subunit vaccines are described in Berman et al., PCT/US91/02250 (published as number WO91/15238 on Oct. 17, 1991). See also, e.g. Hu et al., Nature 328:721-724 (1987) (vaccinia virus-HIV envelope recombinant vaccine); Arthur et al., J. Virol. 63(12): 5046-5053 (1989) (purified gp120); and Berman et al., Proc. Natl. Acad. Sci. U.S.A. 85:5200-5204 (1988) (recombinant envelope glycoprotein gp120).
  • gp120 Numerous sequences for gp120 are known.
  • the sequence of gp120 from the IIIB substrain of HIV-1 LAI referred to herein is that determined by Muesing et al., "Nucleic acid structure and expression of the human AIDS/lymphadenopathy retrovirus, Nature 313:450-458 (1985).
  • the sequences of gp120 from the NY-5, Jrcsf, Z6, Z321, and HXB2 strains of HIV-1 are listed by Myers et al., "Human Retroviruses and AIDS; A compilation and analysis of nucleic acid and amino acid sequences," Los Alamos National Laboratory, Los Alamos, N. Mex. (1992).
  • the sequence of the Thai isolate A244 is provided by McCutchan et al., "Genetic Variants of HIV-1 in Thailand,” AIDS Res. and Human Retroviruses 8:1887-1895 (1992).
  • the MN 1984 clone is described by Gurgo et al., "Envelope sequences of two new United States HIV-1 isolates," Virol. 164: 531-536 (1988).
  • MN, MN-rgp120, the MN clone or isolate refers to MN GNE .
  • the MN GNE amino acid sequence is Sequence ID NO:41.
  • Oligonucleotide sequences encoding gp120 polypeptides from breakthrough isolates of vaccine trials using MN-rgp120 and the encoded gp120 polypeptides are provided.
  • Use of the gp120 polypeptides from one or more of the isolates in a subunit vaccine, usually together with MN-rgp120, can provide protection against HIV strains that are sufficiently different from the vaccine strain (e.g.; MN-rgp120) that the vaccine does not confer protection against those strains.
  • Antibodies induced by the polypeptides are also provided.
  • FIGS. 1A-1G illustrate the kinetics of antibody response to MN-rgp120 in vaccinees infected with HIV-1.
  • Sera were collected at the time points indicated and assayed for antibodies reactive with MN-rgp120 (open circles) or a synthetic peptide derived from the V3 domain of MN-rgp120 (closed circles).
  • Arrows indicate dates of injection. Plus sign indicates the first time HIV-1 infection was detected. Shaded area indicates data collected after HIV-1 infection.
  • Data from vaccinee C6 is shown in FIG. 1A; C8 in FIG. 1B; C7, FIG. 1C; C11, FIG. 1D; C10, FIG. 1E; C17, FIG. 1F; and C15, FIG. 1G.
  • FIGS. 2A-2G illustrate the kinetics of CD4 blocking antibody response in vaccinees infected with HIV-1.
  • Sera were collected at the time points indicated and assayed for antibodies able to block the binding of [ 125 I]-labeled MN-rgp120 to cell surface CD4.
  • Arrows indicate dates of injection. Plus sign indicates the first time HIV-1 infection was detected. Shaded area indicates data collected after HIV-1 infection.
  • Data from vaccinee C6 is shown in FIG. 2A; C8 in FIG. 2B; C7, FIG. 2C; C11, FIG. 2D; C10, FIG. 2E; C17, FIG. 2F; and C15, FIG. 2G.
  • FIGS. 3A-3J illustrate predicted amino acid sequences of envelope glycoproteins (gp120) from breakthrough viruses.
  • Proviral DNA sequences were amplified by PCR from PBMCs and cloned into the PRK5 expression plasmid. Two clones from each infected vaccinee were sequenced from double stranded plasmid DNA. Sequence numbering is with reference to the initiator methionine residue of gp120. For the purpose of comparison, the sequences shown begin at amino acid 12 of the mature, fully processed, envelope glycoproteins (corresponding to position 41 of the gp120 open reading frame).
  • Shaded areas indicate sequences at neutralizing epitopes, dark boxes indicate polymorphisms thought to be important for the binding of virus neutralizing MAbs reactive with MN-rgp120. conserveed (C) regions and variable (V) regions are indicated above the sequences. Boxes indicate sequence homologies and polymorphisms.
  • FIG. 4 illustrates immunoprecipitation of recombinant gp120 prepared from breakthrough viruses.
  • Recombinant gp120s from the seven breakthrough viruses were prepared by transient transfection of 293s cells. Cells were metabolically labeled with 35 S methionine and growth conditioned cell culture supernatants were immunoprecipitated with polyclonal antisera to MN-rgp120. Immunoprecipitates were resolved by SDS-PAGE and visualized by autoradiography.
  • C8 lanes a and b correspond to clones C8.3 and C8.6;
  • C6 lanes a and b correspond to clones C6.1 and C6.5;
  • C7 lanes a and b correspond to clones C7.2 and C7.10;
  • C17 lanes a and b correspond to C17.1 and C17.3;
  • C11 lanes a and b correspond to clones C11.5 and C11.7;
  • C10 lanes a and b correspond to clones C10.5 and C10.7;
  • C15 lanes a and b correspond to clones C15.2 and C15.3.
  • FIGS. 5A-5D illustrate binding of monoclonal antibodies to recombinant gp120 from breakthrough viruses.
  • Growth-conditioned cell culture supernatants were collected from 293s cells transiently transfected with plasmids directing the expression of breakthrough virus envelope glycoproteins.
  • the relative rgp120 concentrations were determined by ELISA using MAb 5B6 specific for the HSV-1 glycoprotein D flag epitope at the amino terminus of all of the rgp120 variants described herein.
  • the resulting rgp120 preparations were captured onto wells of microtiter plates coated with a polyclonal antibody specific for a conserved sequence in the C-terminus of gp120.
  • FIG. 5A binding by MAb (5B6) specific for the HSV-1 glycoprotein D flag epitope
  • FIG. 5B binding by MAb (1034) against the V3 domain of MN-rgp120
  • FIG. 5C binding by MAb (50.1) raised against a synthetic peptide corresponding to the V3 domain of MN-rgp120
  • FIG. 5D binding by a human MAb (15e) known to block the binding of gp120 to CD4.
  • FIG. 6 depicts the mature envelope glycoprotein (gp120) from the MN clone of the MN strain of HIV-1 (SEQ. ID. NO:41). Hypervariable domains are indicated in bold, and the V and C regions are indicated (according to Modrow et al., J. Virology 61(2) :570 (1987). Potential glycosylation sites are marked with a (*).
  • the present invention provides gp120 polypeptides from breakthrough isolates of HIV vaccine trials. Novel oligonucleotide sequences encoding gp120 from breakthrough isolates which can be used to express gp120 are also provided. Use of gp120 polypeptides from one or more of the isolates in a subunit vaccine, usually together with MN-rgp120, can provide protection against HIV strains that are sufficiently different from the vaccine strain (e.g.; MN-rgp120) that the vaccine does not confer protection against those strains.
  • the vaccine is based on the use of the MN-rgp120 polypeptide (Sequence ID No:41) and gp120 polypeptides from MN-like viruses that include neutralizing epitopes that are not present in the initial vaccine strain, and are sufficiently different from those of the vaccine strain, to have been able to cause HIV-1 infections in MN-rgp120 vaccinated individuals (i.e.; to result in breakthrough infections).
  • Use of the initial vaccine strain empirically determines the viruses present in the population that contain additional neutralizing epitopes sufficiently different from those of the vaccine strain to escape protection induced by the vaccine strain.
  • an initial representative gp120 polypeptide in a vaccine acts as a sieve so that viruses that are not effectively protected against by the vaccine strain breakthrough the vaccine, empirically resulting in determination of additional strains in a given geographic region that are not protected against by the initial vaccine strain.
  • Use of gp120 from those breakthrough isolates complements the vaccine isolate by providing additional neutralizing epitopes not present in the initial vaccine strain, therefore creating a more complete vaccine that confers protection against multiple different virus strains in the region.
  • Prior HIV-1 vaccine strategies were based on selection of appropriate candidate vaccine polypeptides based on homology alignment studies. However, since some of the neutralizing epitopes are conformation-dependent and the location of all of these epitopes is not known, this approach necessarily cannot determine all of the neutralizing epitopes that should be included in a vaccine for a particular region. In contrast, the present approach uses a selected representative strain and empirically determines strains that are sufficiently different and therefore breakthrough the barrier of protection provided by the initial vaccination program. Those strains can be included in the vaccine to confer more complete protection from HIV strains in the region. In addition, those strains can be used alone to confer protection against the breakthrough virus.
  • the invention comprises a vaccine containing a first HIV gp120 polypeptide sequence and a breakthrough isolate HIV gp120 polypeptide sequence from a vaccinee vaccinated with a vaccine including the first HIV gp120 polypeptide sequence, the HIV gp120 polypeptide sequences being in a suitable carrier. Fragments of one or both HIV gp120 polypeptide sequences can be substituted for one or both of the corresponding HIV gp120 polypeptide sequences.
  • the first gp120 polypeptide sequence contains neutralizing epitopes found in one or more gp120 polypeptides present in isolates from the geographical region where the initial vaccine (i.e., the vaccine that gives rise to the breakthrough isolate) is administered. More preferably, the first gp120 polypeptide sequence contains at least one of the more common neutralizing epitopes for the region, and most preferably the first gp120 polypeptide sequence contains at least one of the three most common neutralizing epitopes.
  • gp120 polypeptide sequences suitable for use as the first gp120 polypeptide sequence include gp120 MN, the Thai isolate A244 sequence (hereinafter "gp120 A244"), gp120 MN-GNE6 (Sequences ID NOs:43 and 44; also known in the art as “gp120 GNE6"), and gp120 MN-GNE8 (Sequence ID NO:46; also known in the art as "gp120 GNE8”), and the like.
  • gp120 MN, gp120 MN-GNE6, and gp120 MN-GNE8 are especially preferred for use as the first gp120 polypeptide sequence in initial vaccines for North America.
  • gp120 A244 is especially preferred for use as the first gp120 polypeptide sequence in initial vaccines for Thailand.
  • the vaccine includes two different (i.e., first and second) gp120 polypeptide sequences, or fragments thereof, in combination with a breakthrough isolate HIV gp120 polypeptide sequence.
  • the latter can be from a vaccinee vaccinated with either or both of the first and second HIV gp120 polypeptide sequences.
  • Exemplary vaccines include those containing combinations of gp120 MN, gp120 A244, gp120 MN-GNE6 (Sequence ID NOs:43 and 44), and gp120 MN-GNE8 (Sequence ID NO:46). Combinations of gp120 MN and gp120 A244 or gp120 MN-GNE8 (Sequence ID NO:46) with a breakthrough isolate HIV gp120 polypeptide sequence are especially preferred.
  • the breakthrough isolate HIV gp120 polypeptide sequence can be an HIV gp120 polypeptide sequence selected from the group consisting of Sequence ID NOs:2, 5, 8, 10, 12, 16, 19, 23, 25, 28, 31, 33, 36, and 39, and fragments thereof.
  • subunit vaccine is used herein, as in the art, to refer to a viral vaccine that does not contain virus, but rather contains one or more viral proteins or fragments of viral proteins.
  • multivalent means that the vaccine contains gp120 from at least two HIV isolates having different amino acid sequences.
  • breakthrough isolate or "breakthrough virus” is used herein, as in the art, to refer to a virus isolated from a vaccinee.
  • amino acid sequence amino acid sequence
  • polypeptide sequence amino acid sequence
  • polypeptide amino acid sequence
  • oligonucleotide amino acid sequence
  • the gp120 polypeptides of this invention correspond to the amino acid sequences of seven breakthrough isolates which are illustrated below in Table 1.
  • a polypeptide of this invention includes an HIV gp120 amino acid sequence illustrated in Table 1 (Sequence ID NOs:1, 4, 7, 9, 11, 15, 18, 22, 24, 27, 30, 32, 35, and 38) and fragments thereof.
  • the polypeptides of this invention can include fused sequences from two or more HIV gp120 or gp160 amino acid sequences.
  • the polypeptide can also be joined to another viral protein, such as a flag epitope amino acid sequence.
  • flag epitope is used herein, as in the art, to denote an amino acid sequence that includes an epitope recognized by a monoclonal antibody.
  • Flag epitopes facilitate using single monoclonal antibody affinity purification of a plurality of different recombinant proteins, each having the flag epitope recognized by the monoclonal antibody. Numerous amino acid sequences can function as flag epitopes.
  • the N-terminal sequences of Herpes Simplex Virus Type 1 (HSV-1) glycoprotein D (gD-1) is conveniently used as the flag epitope and its use is described in detail in the examples.
  • the flag epitope is conveniently fused to the N terminus of the HIV gp120 polypeptide sequence.
  • monoclonal antibodies that recognize neutralizing epitopes in the rgp120 sequences can be used to affinity purify the amino acid sequences, and a flag epitope can be omitted.
  • various signal sequences can be joined to a polypeptide of this invention.
  • rgp120 is secreted to some extent in HIV cultures, the amount of the envelope glycoprotein released from (secreted by) the host cells varies widely from strain to strain.
  • Various signal sequences can be introduced into the polypeptide by joining a nucleotide sequence encoding the signal sequence to the nucleotide sequence encoding the rgp120 to facilitate secretion of rgp120 from the cells.
  • Chiron HIV gp120 polypeptides include a signal sequence from tissue plasminogen activator (TPA) that provides good secretion of rgp120.
  • TPA tissue plasminogen activator
  • Additional signal sequences are well known and include the N-terminal domain of murine leukemia virus surface protein gp70 described by Kayman et al., J. Virol. 68:400-410 (1984).
  • Table 1 illustrates the nucleotide and deduced amino acid sequences for two clones of each the seven breakthrough isolates of this invention.
  • the clones are: C6.1; C6.5; C8.3; C8.6; C15.2; C15.3; C7.2; C7.10; C11.5; C11.7; C10.5; C10.7; C17.1; and C17.3. These sequences are SEQ. ID. NOs:1-40.
  • the amino acid sequence for MN and the nucleotide and deduced amino acid sequences for MN-GNE6 and MN-GNE8 are illustrated in the sequence listing hereinafter. In the listing for MN-GNE6, a stop codon appears at amino acid residue position 51. This stop codon can be replaced with a codon encoding the corresponding amino acid from MN or MN-GNE8 or another isolate.
  • FIG. 3 shows the alignment of the amino acid sequences of the clones of each of the seven isolates. Corresponding residues from various clones are in boxes. In the figure, the amino acid sequences are aligned against MN-rgp120 (SEQ. ID. NO:41).
  • a gp120 polypeptide of this invention has the same amino acid sequence as the sequence of one of the breakthrough isolates.
  • the amino acid sequence is truncated, as described in detail hereinafter.
  • a gp120 polypeptide sequence of this invention contains a substitution, insertion, or deletion (alteration) of one or more amino acids in the sequence of a breakthrough isolate.
  • a gp120 polypeptide of this invention will include alterations in the amino acid sequence of a breakthrough isolate that do not alter the polypeptide's ability to induce the same neutralizing antibodies as the amino acid sequence of the isolate.
  • substitutions in the amino acid sequence of a gp120 polypeptide of this invention are conservative substitutions, particularly for amino acid residues in the V2, V3, and C4 domains of gp120, which domains contain neutralizing epitopes.
  • nonconservative substitutions, particularly in domains that do not contain neutralizing epitopes are contemplated.
  • amino acids can be divided into the following groups: positively charged residues (K, R and H); negatively charged residues (D and E); amides (N and Q); aromatics (F, Y, and W); hydrophobics (P, G, A, V, L, I, and M); and uncharged residues (S and T). Usually, residues within a group are replaced with another member of the group.
  • critical amino acid residues in the V2, V3, and C4 domains of gp120 are identical to the corresponding residues in a breakthrough isolate sequence.
  • Critical amino acid residues in the V2, V3, and C4 domains of gp120 are described in the experimental section.
  • all amino acid residues in the V2, V3, and C4 domains of gp120 are identical to corresponding residues in a breakthrough isolate sequence.
  • the present invention also provides novel oligonucleotides encoding gp120 from the breakthrough isolates which can be used to express gp120.
  • An oligonucleotide of this invention encodes a polypeptide of this invention.
  • the oligonucleotide can be DNA or RNA, usually DNA.
  • numerous nucleotide sequences can encode the same amino acid sequence due to the degeneracy of the genetic code, conveniently, the oligonucleotides of this invention include a nucleotide sequence of a breakthrough isolate as illustrated in Table 1 (Sequence ID NOs:1, 4, 7, 9, 11, 15, 18, 22, 24, 27, 30, 32, 35, and 38).
  • an oligonucleotide of this invention is less than about 5 kilobases (kb), preferably less than about 3 kb.
  • the oligonucleotide can be inserted into a transcription unit.
  • the transcription unit can be inserted into a plasmid for production of cell lines, inserted into a virus (e.g.; vaccinia) or can be used directly as a DNA vaccine. Suitable transcription units for production of vaccine proteins are well known.
  • a preferred expression vector, designated psvI6B5, is illustrated in Sequence ID NO:45.
  • the vector includes an HSV-1 gD1 signal sequence joined to a linker sequence.
  • the gp120 nucleotide sequence to be expressed starts with the Kpn I site of the gene. Since all gp120 or gp160 sequences contain this site, any gp120 nucleotide sequence can be analogously inserted into the vector and expressed.
  • the vector ends with a polyA tail from SV40.
  • the oligonucleotides of this invention can also be used in diagnostics to detect HIV isolates.
  • the oligonucleotide or a portion thereof encoding a neutralizing epitope can be used in branched chain DNA diagnostics or as a probe in in situ hybridization studies.
  • a gp120 polypeptide of this invention from a selected breakthrough isolate(s) in a suitable carrier is used to make a subunit vaccine.
  • the polypeptide can be used alone, but is generally administered in a multivalent subunit vaccine that includes gp120 MN.
  • the vaccine generally includes the MN polypeptide (hereinafter, MN-rgp120).
  • MN-rgp120 MN polypeptide
  • the vaccine usually includes about 3 to about 5 different gp120 polypeptides, but 30 or more different gp120 polypeptides can be used.
  • gp120 polypeptides for use in a vaccine is well known and is described hereinafter. With the exception of the use of the selected HIV isolate, the gp120 subunit vaccine prepared in the method does not differ from gp120 subunit vaccines of the prior art.
  • gp120 at the desired degree of purity and at a sufficient concentration to induce antibody formation is mixed with a physiologically acceptable carrier.
  • a physiologically acceptable carrier is nontoxic to a recipient at the dosage and concentration employed in the vaccine.
  • the vaccine is formulated for injection, usually intramuscular or subcutaneous injection. Suitable carriers for injection include sterile water, but preferably are physiologic salt solutions, such as normal saline or buffered salt solutions such as phosphate-buffered saline or ringer's lactate.
  • the vaccine generally contains an adjuvant.
  • Useful adjuvants include QS21 (Quillaja saponaria, commercially available from Cambridge Biotech, Worcester, Mass.), which stimulates cytotoxic T-cells, and alum (aluminum hydroxide adjuvant). Formulations with different adjuvants which enhance cellular or local immunity can also be used.
  • immunopotentiators such as cytokines can be included in the vaccine.
  • suitable immunopotentiating cytokines include interleukins, such as interleukin-2 (IL-2) and interleukin-12 (IL-12), and tumor necrosis factor-alpha (TNF- ⁇ ).
  • excipients that can be present in the vaccine include low molecular weight polypeptides (less than about 10 residues), proteins, amino acids, carbohydrates including glucose or dextrans, chelating agents such as EDTA, and other excipients that stabilize the protein or inhibit growth of microorganisms.
  • the vaccine can also contain other HIV proteins.
  • gp41 or the extracellular portion of gp41 or HIV-1 core proteins such as P24, P17, and P55 can be present in the vaccine.
  • the amino acid sequence of gp41 is more conserved than that of gp120, gp41 contains neutralizing epitopes.
  • any gp41 present in the vaccine is from an HIV isolate present in the vaccine.
  • gp160 from an isolate used in the vaccine can replace gp120 in the vaccine or be used together with gp120 from the isolate.
  • gp160 from a different isolate than those in the vaccine can additionally be present in the vaccine.
  • Vaccines according to the invention can also contain one or more soluble gp120 polypeptide sequences, or fragments thereof, in combination with an engineered virus specifically designed to express proteins that induce a cytotoxic T-cell response.
  • engineered viruses are derived from, for example, Canary Pox virus, vaccinia viruses, attenuated human herpes viruses (such as, e.g., herpes simplex viruses), and Varicella Zoster.
  • Exemplary engineered viruses are modified to express any HIV protein capable of inducing a cytotoxic T-cell response, such as those described above.
  • viruses can be engineered to express one or more gp120 polypeptide sequences of the invention, or fragments thereof, and used in vaccines with or without soluble gp120 polypeptide sequences.
  • Vaccine formulations generally include a total of about 300 to 600 ⁇ g of gp120, conveniently in about 1.0 ml of carrier. Preferred formulations include use of twice the weight of a gp120 polypeptide in twice as600 ⁇ g alum. However, formulations having smaller amounts (e.g.; 50 ⁇ g per dose) are also used, generally with alum or other adjuvants.
  • the amount of gp120 for any isolate present in the vaccine will vary depending on the immunogenicity of the gp120. For example, gp120 from some strains of HIV may be less immunogenic than gp120 from the MN strain (Sequence ID NO:41).
  • the vaccine includes gp120 from the MN and a strain of this invention at concentrations of about 300 ⁇ g per strain in about 1.0 ml of carrier.
  • the vaccine includes gp120 from about 30 isolates, about 10 to about 50 ⁇ g can be used.
  • the vaccines of this invention are administered in the same manner as prior art HIV gp120 subunit vaccines.
  • the vaccines are generally administered at 0, 1, and at 6, 8 or 12 months, depending on the protocol.
  • a preferred protocol includes administration at 0, 1, 6, and 12 months.
  • annual or bi-annual boosts can be administered.
  • neutralizing antibody levels can be assayed and the protocol adjusted accordingly.
  • the vaccine is administered to uninfected individuals.
  • the vaccine can be administered to seropositive individuals to augment immune response to the virus, as with prior art HIV vaccines.
  • DNA encoding the strains of gp120 for the vaccine can be administered in a suitable vehicle for expression in the host. In this way, gp120 can be produced in the infected host, eliminating the need for repeated immunizations. Preparation of gp120 expression vehicles is described hereinafter.
  • the amino acid sequences can also be used alone or in combinations in the same type of formulation for use as an immunogen, to induce antibodies that recognize the isolate(s) present in the immunogen.
  • Immunogens are formulated in the same manner as vaccines and can include the same excipients, etc.
  • Antibodies induced by the immunogens can be used in a diagnostic to detect the HIV strain in the immunogen or to affinity purify the strain.
  • CD4 is the primary cellular receptor for HIV-1, it is not sufficient for entry of HIV-1 into cells.
  • Co-receptors required in conjunction with CD4 have been identified. These co-receptors are members of the chemokine receptor family of seven-transmembrane G-protein coupled receptors. The chemokine superfamily is subdivided into two groups based on the amino terminal cysteine spacing. The CXC chemokines are primarily involved in neutrophil-mediated inflammation, and the CC chemokines tend to be involved in chronic inflammation.
  • At least five CC chemokine receptors designated CC-CKR1-5 (also known in the art as CCR1-5), and at least four CXC chemokine receptors, designated CXC-CKR1-4 (also known as CXCR-1-4), have been identified.
  • CXC-CKR-4 (CXCR-4), which has also been called the alpha-chemokine receptor fusin, serves as an entry cofactor for T-cell-tropic HIV-1 strains.
  • CC-CKR-5 (CC-R5), which has been called beta-chemokine receptor, together with its related family members, such as CC-CKR-2b and CC-CKR3, serve as entry cofactors for macrophage-tropic HIV-1 strains.
  • T-cell-tropic strains can infect primary T-cells and T-cell lines, but not macrophages, whereas macrophage-tropic strains can infect macrophages and primary T-cells, but not T-cell lines.
  • T-cell- and macrophage-tropic strains are discussed more fully in Deng et.
  • T-cell-tropic strains include laboratory isolates, such as IIIB and MN.
  • Macrophage-tropic strains include primary isolates, including but not limited to A244, GNE6, GNE8, and breakthrough viruses from vaccinees immunized with gp120-based vaccines.
  • Dual-tropic strains can use both types of co-receptors, entering cells via CXC-CKR-4 or via one or more CC-CKR family members, preferably CC-CKR-5, CC-CKR-2b, or CC-CKR-3.
  • T-cell tropic and macrophagetropic HIV-1 strains are believed to provide a unifying explanation of the differences in cell tropism between viral strains, the resistance to HIV-1 infection by many CD4-transfected nonprimate cells, and the HIV-1 infection resistance of a portion of the human population.
  • a vaccine containing (1) a first gp120 polypeptide sequence, or fragment thereof, from a macrophage-tropic HIV-1 strain and/or a second gp120 polypeptide sequence, or fragment thereof, from a T-cell tropic strain, in combination with (2) a breakthrough isolate HIV gp120 polypeptide sequence, or fragment thereof, from a vaccinee vaccinated with the first and/or second HIV gp120 polypeptide sequence.
  • the vaccine includes at least two gp120 polypeptide sequences that bind to different chemokine receptors.
  • the vaccine includes first and second gp120 polypeptide sequences that bind to different chemokine receptors.
  • the breakthrough isolate gp120 polypeptide sequence can bind to a different chemokine receptor than the chemokine receptor(s) bound by either or both of the first and second gp120 polypeptide sequence(s).
  • a preferred T-cell tropic strain is a laboratory isolate, most preferably MN.
  • Preferred macrophagetropic viruses for use in the invention are GNE6 and GNE8, which are representative of the breakthrough viruses disclosed herein and differ from MN in that their gp120s induce the formation of antibodies that recognize the gp120 sequences (e.g., the V3 domain) involved in binding to CC chemokine receptors, such as CXC-CKR-5.
  • HIV infection is prevented by administering one or more chemokine receptor-binding gp120 polypeptide sequences, or fragment(s) thereof containing appropriate chemokine receptor-binding domains, in a vaccine, such as those described above.
  • the vaccine also includes one or more CD4-binding gp120 polypeptide sequences or appropriate fragments thereof.
  • Such vaccines induce anti-HIV antibodies that inhibit viral gp120-chemokine receptor or -CD4 binding.
  • such gp120 polypeptides can directly inhibit HIV infection by binding to one or more co-receptors for HIV infection, such as CD4 or a chemokine receptor, thus providing a prophylactic or therapeutic effect in treating HIV infection.
  • gp120 polypeptide sequences useful in this regard contain the T-cell binding (TCB) domain.
  • chemokine receptor-binding gp120 polypeptides are discussed below with regard to the CC chemokine receptor family. However, those skilled in the art recognize that this discussion applies equally to CXC chemokine receptors that act as cofactors in HIV infection.
  • the gp120 polypeptides can be used as a composition containing one or more gp120 polypeptides, as described for use as a vaccine or immunogen.
  • the composition can be administered, prophylactically or therapeutically, to a patient at risk of infection or in need of such treatment using the dosages and routes and means of administration described herein. However, chronic administration may be preferred and dosages can be adjusted accordingly. It is noted that in vivo administration can also induce antibodies that bind viral gp120, further inhibiting virus binding to CC-CKR.
  • the gp120 polypeptides can also be used in screening assays to identify antagonists of CC-CKR.
  • candidate antagonists can be screened for inhibition of binding of gp120 to a CC-CKR CC-CKR receptor that is isolated and attached to a surface (e.g., plastic dish) or recombinantly or naturally expressed on the surface of a cell.
  • Antagonists can either bind gp120 or bind receptor.
  • Preferred candidate antagonists include gp120 compounds, small gp120 peptides (5 to 20 amino acids in length, preferably 7 to 10 amino acids in length) or peptidomimetics of gp120 that bind receptor, monoclonal antibodies that bind gp120, and small organic molecules that bind either gp120 or receptor.
  • the antibodies induced by the gp120 polypeptides can also be used to induce anti-idiotype antibodies that bind CC chemokines. These anti-idiotype antibodies can be screened for binding to an anti-gp120 polypeptide antibody and inhibiting gp120 from binding CC-CKR receptor. Such anti-idiotype antibodies mimic gp120 by binding to CC-CKR receptor.
  • Such antibodies preferably human antibodies, can be obtained in a number of ways, such as human antibodies from combinatorial libraries (e.g., Burton et al. Adv. Immunolo. (1994) 57:191-280).
  • transgenic animals e.g., mice
  • transgenic animals e.g., mice
  • homozygous deletion of the antibody heavy-chain joining region (JH) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production.
  • Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice results in the production of human antibodies upon antigen challenge as described in Jakobovitis et al., Proc. Natl. Acad. Sci. USA 90: 2551 (1993); Jakobovits et al., Nature 362:255-258 (1993); Bruggermann et al., Year in Immuno. 7: 33 (1993).
  • phage display technology as described by McCafferty et al., Nature 348:552-553 (1990) can be used to produce human antibodies and antibody fragments in vitro from immunoglobulin variable (V) domain gene repertoires from unimmunized donors.
  • V domain genes are closed in-frame either into either a major or minor coat protein gene of a filamentous bacteriophage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties.
  • Phage display can be performed in a variety of formats as reviewed by, for example, Johnson, et al., Current Opinion in Structural Biology 3:564-571 (1993).
  • V-gene segments can be used for phage display.
  • Clackson et al., Nature, 352: 624-628 (1991) isolated a diverse array of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. A repertoire of V genes from unimmunized human donors (or embryonic cells) can be constructed. It has been demonstrated that antibodies to a diverse array of antigens (including self-antigens) can be isolated essentially following the techniques described by Marks et al., J. Mol. Biol., 222: 581-597 (1991), or Griffith et al., EMBO J., 12: 725-734 (1993).
  • antibodies that bind CC-CKR can be obtained by screening antibodies or fragments thereof expressed on the surface of bacteriophage in combinatorial libraries or in other systems as described above with a gp120 monoclonal antibody that inhibits gp120 binding to receptor.
  • random or combinatorial peptide libraries can be screened with either a gp120 antibody or the gp120 compounds of the invention.
  • Approaches are available for identifying peptide ligands from libraries that comprise large collections of peptides, ranging from 1 million to 1 billion difference sequences, which can be screened using monoclonal antibodies or target molecules.
  • the power of this technology stems from the chemical diversity of the amino acids coupled with the large number of sequences in a library. See for example, Scott et al., Cur. Open. Biotechnol. 5(1):40-8 (1994); Kenan et al. Trends Biochem. Sci. (1994) 19(2):57-64.
  • the monoclonal antibodies, preferably human monoclonals or fragments thereof, generated as discussed herein, find use in treatment by inhibiting or treating HIV infection or disease progression, as well as in screening assays to identify additional pharmaceuticals.
  • gp120 for a vaccine can be produced by any suitable means, as with prior art HIV gp120 subunit vaccines. Recombinantly-produced or chemically synthesized gp120 is preferable to gp120 isolated directly from HIV for safety reasons. Methods for recombinant production of gp120 are described below.
  • Oligonucleotides encoding gp120 from breakthrough isolates and capable of expressing gp120 can be prepared by conventional means.
  • the nucleotide sequence can be synthesized.
  • another HIV nucleotide sequence encoding gp120 can be used as a backbone and altered at any differing residues as by site-directed mutagenesis.
  • Site-directed mutagenesis is described in Kunkel et al, Proc. Natl. Acad. Sci. (USA) 82:488-492 (1985) and Zoller et al, Nuc. Acids Res. 10:6487-6500 (1982) and is well known.
  • the nucleotide sequence is present in an expression construct containing DNA encoding gp120 under the transcriptional and translational control of a promoter for expression of the encoded protein.
  • the promoter can be a eukaryotic promoter for expression in a mammalian cell.
  • the promoter can be a prokaryotic promoter.
  • a strong promoter is employed to provide high-level transcription and expression.
  • the expression construct can be part of a vector capable of stable extrachromosomal maintenance in an appropriate cellular host or may be integrated into host genomes. Normally, markers are provided with the expression construct which allow for selection of a host containing the construct.
  • the marker can be on the same or a different DNA molecule, desirably, the same DNA molecule.
  • the expression construct can be joined to a replication system recognized by the intended host cell.
  • Various replication systems include viral replication systems such as those from retroviruses, simian virus, bovine papilloma virus, or the like.
  • the construct may be joined to an amplifiable gene, e.g. the DHFR gene, so that multiple copies of the gp120 DNA can be made.
  • Introduction of the construct into the host will vary depending on the construct and can be achieved by any convenient means.
  • a wide variety of prokaryotic and eukaryotic hosts can be employed for expression of the proteins.
  • the gp120 is expressed in mammalian cells that provide the same glycosylation and disulfide bonds as in native gp120.
  • Expression of gp120 and fragments of gp120 in mammalian cells as fusion proteins incorporating N-terminal sequences of Herpes Simplex Virus Type 1 (HSV-1) glycoprotein D (gD-1) is described in Lasky, L. A. et al., 1986 (Neutralization of the AIDS retrovirus by antibodies to a recombinant envelope glycoprotein) Science 233: 209-212 and Haffar, O. K. et al., 1991 (The cytoplasmic tail of HIV-1 gp160 contains regions that associate with cellular membranes.) Virol. 180:439-441, respectively.
  • a preferred method for expressing gp120 is described in the examples. In the examples, a heterologous signal sequence was used for convenient expression of the protein. However, the protein can also be expressed using the native signal sequence.
  • an isolated, purified gp120 polypeptide having one of the amino acid sequences illustrated in Table 1 can be produced by conventional methods.
  • the proteins can be chemically synthesized.
  • the proteins are expressed in mammalian cells using an expression construct of this invention.
  • the expressed proteins can be purified by conventional means. A preferred purification procedure is described in the examples.
  • the present invention also provides gp120 fragments that are suitable for use in inducing antibodies for use in a vaccine formulation.
  • a truncated gp120 sequence as used herein, is a fragment of gp120 that is free from a portion of the intact gp120 sequence beginning at either the amino or carboxy terminus of gp120.
  • a truncated gp120 sequence of this invention is free from the C5 domain.
  • the C5 domain of gp120 is a major immunogenic site of the molecule.
  • antibodies to the region do not neutralize virus. Therefore, elimination of this portion of gp120 from immunogens used to induce antibodies for serotyping is advantageous.
  • the truncated gp120 sequence is additionally free from the carboxy terminal region through about amino acid residue 453 of the gp120 V5 domain.
  • the portion of the V5 domain remaining in the sequence provides a convenient restriction site for preparation of expression constructs.
  • a truncated gp120 sequence that is free from the entire gp120 V5 domain is also suitable for use in inducing antibodies.
  • portions of the amino terminus of gp120 can also be eliminated from the truncated gp120 sequence.
  • the truncated gp120 sequence can be free from the gp120 signal sequence.
  • the truncated gp120 sequence can be free from the carboxy terminus through amino acid residue 111 of the gp120 C1 domain, eliminating most of the C1 domain but preserving a convenient restriction site.
  • the portion of the C1 domain through the V2 cysteine residue that forms a disulfide bond can additionally be removed, so that the truncated gp120 sequence is free from the carboxy terminus through amino acid residue 117 of the gp120 C1 domain.
  • the truncated gp120 sequence is free from the amino terminus of gp120 through residue 111 of the C1 domain and residue 453 through the carboxy terminus of gp120.
  • the truncated gp120 sequences can be produced by recombinant engineering, as described previously. Conveniently, DNA encoding the truncated gp120 sequence is joined to a heterologous DNA sequence encoding a signal sequence.
  • Specimen collection from human volunteers Blood was collected from MN-rgp120-immunized individuals who were infected with HIV-1 while participating in Phase I (NIH Protocol AVEG 016) and Phase II (NIH Protocol AVEG 201) HIV-1 vaccine trials sponsored by the National Institutes of Health (NIH).
  • the demographics of the subjects in the study, and the study design have been described in McElrath; Seminars in Cancer Biol. 6:1-11 (1995); McElrath et al.; Abstracts from Eighth Annual Meeting of the National Cooperative Vaccine Development Groups for AIDS. Bethseda, Md. 216 (1996).
  • Specimens were obtained according to an informed consent protocol approved by the institutional review boards of the participating institutions.
  • the time of HIV-1 infection is specified with regard to data provided by the NIH AIDS Vaccine Evaluation Network where PCR (RNA) and/or serologic assays were used to detect HIV-1 infection.
  • PBMCs Peripheral blood mononuclear cells
  • PBMCs were washed twice in phosphate-buffered saline and suspended at a cell density of 6 ⁇ 10 6 cells/ml in PCR lysis buffer (50 mM KCl, 10 mM Tris (pH 8.4), 2.5 mM MgCl 2 , 0.1 mg/ml gelatin (Sigma), 0.45% NONIDET P40 detergent, 0.45% TWEEN 20 detergent (both detergents are commercially available from United States Biochemical Corp.) and 0.06 mg/ml Proteinase K (Gibco BRL) to lyse the cells.
  • the lysate was incubated at 50-60° C. for 1 hour, followed by inactivation of the Proteinase K at 95° C. for 10 minutes. Lysates were shipped frozen and stored at -70° C. until use.
  • PCR Polymerase chain reaction
  • the PCR profile was as follows: 2 cycles having 1 minute at 55° C., 2.5 minutes at 72° C. and 1 minute at 94° C., followed by 28 cycles with 30 seconds at 55° C., 2.5 minutes at 72° C. and 45 seconds at 94° C., and an extension step at 72° C. for 5 minutes.
  • JM11A (5'-ctcgag-CTCCTGAAGACAGTCAGACTCATCAAG at position 6048-6074)
  • SEQ. ID. NO:48 in the forward direction [Kusumi et al.; J. Virol. 66:875 (1992)] combined with 120.os.R (5'-ggtctagaagctttaGCCCATAGTGCTTCCTGCTGCT-CC at position 7836-7859) (SEQ. ID. NO:49) in the reverse direction.
  • the internal nested primers were 120.BX.F (5'-gggcggatcctcgaGGTACCTGTRTGGAAAGAAGCA at position 6389-6410; R: A or G) (SEQ. ID. NO:50) and 120.is.R (5'-ggtctagaagctttaTGCTCCYAAGAACCCAAGGAACA at position 7819-7841; Y: T or C) (SEQ. ID. NO:51). Heterologous primer sequences are shown in lower case letters.
  • HIV-1 envelope glycoprotein gp120 sequences were cloned and expressed as chimeric genes and fusion proteins, where the signal sequence and 27 amino acids from the mature N terminus of herpes simplex virus type 1 (HSV-1) were fused to the N-terminal sequences of the gp120 genes, corresponding to amino acid 13 of the mature gp120 sequence.
  • HSV-1 herpes simplex virus type 1
  • PCR products containing gp120 sequences from the breakthrough specimens were cloned into pRK5 expression plasmid as chimeric genes using combinations of restrictions sites engineered into the heterologous PCR primer tails and the Xho I site engineered into the N-terminal sequence of HSV-1 gD.
  • the resulting double-stranded DNA was sequenced with Sequenase and the dGTP Reagent Kit (United States Biochemical Corp.). Sequences from glycoprotein D were provided to enhance expression and to provide a flag epitope to facilitate protein analysis, as described in Berman et al.; J. Virol. 7:4464-9 (1992); Nakamura et al.; AIDS and Human Retroviruses 8:1875-85 (1992); and Nakamura et al.; J. Virol. 67:6179-91 (1993).
  • isolated DNA fragments generated by the PCR reaction were ligated into a plasmid (pRK.gD-5, pRKgDstop) designed to fuse the gp120 fragments, in frame, to the 5' sequences of the glycoprotein D (gD) gene of Type 1 Herpes Simplex Virus (gD-1)and the 3' end to translational stop codons.
  • the fragment of the gD-1 gene encoded the signal sequence and 25 amino acids of the mature form of HSV-1 protein.
  • chimeric genes fragments were cloned into the pRK5 expression plasmid (Eaton et al., Biochemistry 291:8343-8347 (1986)) that contained a polylinker with cloning sites and translational stop codons located between a cytomegalovirus promotor and a simian virus 40 virus polyadenylation site.
  • the resulting plasmids were transfected into the 293s embryonic human kidney cell line (Graham et al., J. Gen. Virol. 36:59-77 (1977)) using a calcium phosphate technique (Graham et al., Virology 52:456-467 (1973)).
  • Growth conditioned cell culture media was collected 48 hr after transfection, and the soluble proteins were detected by ELISA or by specific radioimmunoprecipitation where metabolically labeled proteins from cell culture supernatants were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (PAGE) and visualized by autoradiography as described in Berman et al., J. Virol. 63:3489-3498 (1989) and Laemmli, Nature 227:680-685 (1970).
  • PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
  • Serologic assays Sera were assayed for antibodies to rgp120, antibodies to synthetic gp120 V3 domain peptides corresponding to sequences from the gp120 V3 domain, and antibodies able to inhibit the binding of MN-rgp120 to cell surface CD4 using serologic assays described in Berman et al.; J. Virol. 7:4464-9 (1992); Nakamura et al.; AIDS and Human Retroviruses 8:1875-85 (1992); and Nakamura et al.; J. Virol. 67:6179-91 (1993). Endpoint titers of antibody binding to gp120 and V3 peptides were determined using three fold-serial dilutions of sera.
  • the endpoint dilution titer was defined as the last dilution that produced an optical density value that was two times higher than the mean of the optical densities of 1:50 diluted, pooled, normal human sera.
  • Antibody titers were calculated by a computer program that interpolated values between antibody dilutions. The inter-assay coefficient of variation of positive control standard sera was 35%.
  • Binding of monoclonal antibodies to rgp120 from breakthrough viruses An ELISA similar to that described by Moore et al.; AIDS 3:155-63 (1989) was used to measure the binding of various monoclonal antibodies (MAbs) to rgp120s from breakthrough viruses. Briefly, Nunc-Immuno plates (Maxisorp, certified) were coated (100 ⁇ l at 5 ⁇ g/ml in PBS at 4° C. overnight) with an affinity-purified sheep polyclonal antiserum to a peptide at the C terminus of gp120 (D7324, International Enzymes, Fallbrook, Calif.).
  • the plates were washed 3 times and incubated with 100 ⁇ l of a horseradish peroxidase-conjugated second antibody (goat anti-mouse or anti-human IgG, Cappel) for 1 hour at room temperature. After 3 washes the plates were developed and the OD 492 (optical density at 492 nm) read in a plate reader. Growth conditioned cell culture supernatants were normalized by dilution based on binding by MAb 5B6 which is specific for HSV-1 glycoprotein D fusion protein.
  • Virus neutralization assays The ability of vaccinee sera to inhibit infection of MT4 cells by HIV-1 MN was measured in a cytopathicity assay where cell viability was quantitated using a calorimetric indicator dye, as described in Robertson et al.; J. Virol. Methods 20:195-202 (1988). Briefly, a virus stock of HIV-1 MN (obtained from Dr. Michael Norcross, U.S. Food and Drug Administration) was prepared as the clarified supernatant from chronically infected H9/HIV-1 MN cell culture. H9 cells chronically infected with HIV-MN were pelleted and resuspended in one-tenth the original volume of medium. Cell-associated virus was released by the mechanical shearing effects of rapid vortexing of the cells as described in Wrin et al.; J. Virol. 69:39-48 (1995).
  • Virus neutralization endpoints were quantitated by measurement of OD at 570-650 nm, and then the endpoint titers were calculated as the reciprocal of the antiserum dilution giving a signal that was two-fold above the control signal with unprotected (killed) cells. These titers were typically twice those calculated at 50% protection.
  • Antibody response to gp120 in vaccinated individuals The magnitude and specificity of the antibody response to MN-rgp120 was measured by ELISA in all infected individuals throughout the course of the immunization regime (FIG. 1). Five of the seven subjects exhibited normal antibody response kinetics that included a small but reproducible primary response (1:100-1:2,000) and a strong secondary (booster) response (titters ranging from 1:7,000-1:32,000), and antibody responses following third and fourth injections that were similar or marginally higher than those achieved after the second immunization (FIG. 1, Table 3).
  • the antibody response observed in C7 was unusual in that no antibodies were detectable after the primary injection and a titer of only 1:350 was detected after the second injection. It thus appeared that C7 did not respond to the primary immunization, and that the antibody response obtained after the second injection represented a primary immune response. Consistent with this hypothesis, the third injection elicited a titer of only 1:9,707, typical of those normally seen after two immunizations.
  • Antibody titers to the V3 domain were measured to a synthetic V3 domain peptide of MN-rgp120 containing the principal neutralizing determinant (PND).
  • PND principal neutralizing determinant
  • Five of the seven subjects developed good V3 titers (1:400 to 1:4000) after the second immunization, however two subjects (C7 and C15) required three immunizations before developing significant tiers (FIGS. 1C and 1G).
  • the peak V3 titers in some individuals e.g. C11, C10, C17 appeared to decline with each successive immunization (FIGS. 1D, 1E, and 1F).
  • CD4 Inhibition titers Antibodies that block the binding of gp120 to CD4 represent a heterogeneous class of virus neutralizing antibodies. Some are known to bind to the C4 domain of gp120 [Nakamura et al.; J. Virol. 67:6179-91 (1993); Anderson et al.; J. Infect. Dis. 160:960-9 ((1989)], and some are known to recognize conformation dependent discontinuous epitopes [Berman et al.; J. Virol. 7:4464-9 (1992); Nakamura et al.; J. Virol.
  • CD4 blocking titers were detected in all seven of the vaccinees prior to infection (FIG. 2) with peak titers that ranged from 1:10-1:300. At the last time point prior to infection, the CD4 titers in five of the seven vaccinees was low (1:30 or less).
  • CD4 blocking titer of about 1:300 prior to infection (FIG. 2F).
  • CD4 blocking titer of about 1:300 prior to infection (FIG. 2F).
  • Large increases in CD4 blocking titers (1:100-1:1,000) were seen in five of the seven subjects after HIV-1 infection.
  • These included vaccinees C6, C7, C8, C10, and C11.
  • virus neutralizing activity The virus neutralizing activity of antisera from MN-rgp120-immunized subjects was measured using a calorimetric assay that measured the viability of MT-4 cells after incubation with antibody treated virus (HIV-1 MN ). Since the actual date of infection was not known for any of the breakthrough infections, and serum samples were collected infrequently, the magnitude of the neutralizing antibody response at the time of infection is not known for any of the vaccinees.
  • the serum sample closest to the time of infection was that obtained from C7, where a neutralizing titer of 1:15 to HIV-1 was present three weeks prior to detection of HIV-1 infection (Table 4). In all other cases, however, the interval between the last injection and the time of infection was 10 to 25 weeks.
  • Subject C11 possessed a neutralizing titer of 1:90 at fourteen weeks prior to detection of HIV-1 and a peak titer of 1:500 following the third immunization.
  • vaccinee C17 had a neutralizing titer of 1:150 fourteen weeks prior to infection and a peak titer of 1:400 at two weeks after the third immunization.
  • Sequences of Viruses To evaluate the similarity of the breakthrough viruses with the vaccine antigen, nucleotide sequences for gp120 from all seven breakthrough viruses were determined. Envelope glycoprotein genes were amplified from proviral DNA using the polymerase chain reaction. Sequences were obtained by direct amplification of DNA from lysates of gradient-purified lymphocytes obtained directly from patient blood without any intermediate tissue culture or amplification step.
  • FIG. 3 A listing of the complete gp120 sequences (two clones per specimen) is provided in FIG. 3. All seven envelope glycoproteins possessed sequences typical of subtype (clade) B viruses. The overall homology with MN-rgp120 ranged from 69-80% (Table 5).
  • viruses C6, C8, and C17
  • MN serotype signature sequence FOG. 3
  • four viruses possessed sequences with radical amino acid substitutions in the PND [IGPGRAW (C7), LGPGSTF (C11), IGPGRVL (C10), and IGPGSAF (C15)] (SEQ. ID. NOs:53-56, respectively), and therefore were classified as "non-MN like" viruses.
  • IGPGRAW C7
  • LGPGSTF C11
  • IGPGRVL C10
  • IGPGSAF C15
  • each of the four "non-MN-like" sequences were rare (Table 6) and were not typical of the most common "non-MN" variants of subtype B viruses [Myers et al.; Retroviruses and AIDS Database, Los Alamos National Laboratory (1992 and 1995)].
  • breakthrough virus V3 domains Like MN-rgp120, the V3 domains of all of the breakthrough viruses were 36 amino acids in length. However, all seven viruses differed from MN-rgp120 with respect to the number of glycosylation sites and with respect to the syncytium-inducing (SI) signature sequence.
  • SI syncytium-inducing
  • MN-rgp120 The sequence of MN-rgp120 is somewhat unusual [Myers et al.; Retroviruses and AIDS Database, Los Alamos National Laboratory (1992 and 1995)] in that it lacks an N-linked glycosylation site at position 306 in the V3 domain. The lack of this glycosylation site does not appear to be antigenically significant since antisera to MN-rgp120 are known to neutralize a variety of viruses (e.g. SF-2, DU6587-5, DU4489-5, CC) that possess a glycosylation site at this position [Berman et al.; J. Virol. 7:4464-9 (1992)].
  • viruses e.g. SF-2, DU6587-5, DU4489-5, CC
  • V3 domain of MN-rgp120 possessed sequence polymorphisms (R at position 311, K at position 324, K at position 328) typical of syncytium inducing viruses [Fouchier et al.; J. Virol. 66:3183-87 (1992)], whereas all seven breakthrough viruses possessed sequences associated with non-syncytium-inducing viruses.
  • Syncytium-inducing viruses have been associated with rapid disease progression [Tersmette et al.; J. Virol.
  • V1 and V2 domains exhibit several common polymorphisms that affect the binding of virus neutralizing antibodies.
  • One such polymorphism occurs at position 171 which is critically important for the binding of murine MAb 1025, whereas residue 187 is important for the binding of MAb several MAbs represented by 1088.
  • the V2 region of the breakthrough viruses showed less variation than expected.
  • the length of the V2 region ranged from 36 amino acids (C7) to 39 amino acids in length, with six of seven viruses containing three N-linked glycosylation sites in this domain.
  • a high degree of polymorphism was found in the V4 region where sequences ranged from 26 (C10) to 33 (C15, C7) amino acids in length and contained either 4 or 5 N-linked glycosylation sites.
  • the antigenic structure of the V3 domain was examined using the 1034 MAb (isolated from mice immunized with MN-rgp120 as described in Nakamura et al.; J. Virol. 67:6179-91 (1993) and the 50.1 MAb (prepared from mice immunized with a synthetic V3 domain peptide as described in Rini et al.; Proc. Natl. Acad. Sci. USA 90:6325-9 (1993). Both MAbs are known to exhibit potent virus neutralizing activity. When binding to the recombinant proteins was examined, the MAb binding to MN-rgp120 was at least 10-fold greater than to any of the breakthrough virus envelope proteins (FIGS. 5B and C).
  • IGPGRAF SEQ. ID. NO:32
  • antibody titers to MN-rgp120 typically decay with a half-time of 2 to 2.5 months [Belshe et al.; JAMA 272(6):475-80 (1994); Berman et al.; AIDS 8:591-601 (1994)], antibody titers would be expected to have decayed at least eight-fold and possibly as much as sixty four-fold at the time of infection. Thus, the lack of a sufficient immune response at the time of infection represents a potential explanation for at least six of the seven breakthrough infections.
  • formulations e.g. novel adjuvants
  • immunization regimes frequently boosting
  • Use of a booster every six months may be advantageous.
  • the other likely explanation for the late infections is the antigenic difference between the vaccine and the breakthrough virus envelope glycoproteins. This explanation is supported by the observation that four of the seven breakthrough viruses possessed envelope glycoproteins that differed from the MN-rgp120 by 25-30% at the amino acid level. Differences of this magnitude have historically [Myers et al.; Retroviruses and AIDS Database, Los Alamos National Laboratory (1992 and 1995)] been associated with inter-subtype variation and far exceeds the average 10-20% variation expected for viruses within the same subtype.
  • candidate vaccines based on the LAI strain of HIV-1 (the prototypic "non-MN-like" subtype B virus), exhibited little or no cross neutralizing activity with subtype B viruses, whereas vaccines that contained the "MN-like-" PND sequence (IGPGRAF) (SEQ. ID. NO:52) exhibited broad cross neutralizing activity. That four of the seven breakthrough viruses possessed envelope glycoproteins with radical amino acid substitutions in the PND is consistent with the explanation that differences in antigenic structure explain some of these infections.
  • V1 epitopes Although far less is known about the V1 epitopes relative to other neutralizing sites, the V1 epitopes appear to be conformation-dependent, and antisera from HIV-1 infected individuals recognize epitopes in the V1 and V2 domains [McKeating et al.; J. Virol. 67:4932-44 (1993); Kayman et al.; J. Virol. 68:400-410 (1994)].
  • the V1 sequence of the virus from C17 is noteworthy because it is smaller and contains fewer N-linked glycosylation sites than that of MN-rgp120 or any of the other breakthrough viruses.
  • the envelope glycoproteins from C11 and C6 are noteworthy because they are significantly larger and contain more glycosylation sites than MN-rgp120 or the other breakthrough viruses.
  • MN-rgp120 is less than 100% effective against HIV-1 infection. Based on previous in vitro and in vivo studies with MN-rgp120, protection from natural HIV-1 infection in humans is expected to depend on a threshold concentration of virus-neutralizing antibodies, and antigenic similarity between the vaccine immunogen and the challenge virus.
  • viruses and viral glycoproteins derived from breakthrough infections may provide an important means to streamline the process of identifying new antigens for inclusion into a multivalent vaccine.
  • Recombinant viral glycoproteins prepared from breakthrough viruses possess antigenic structures that are significantly different from MN-rgp120, and are be representative of viruses currently being transmitted.
  • combining rgp120 from breakthrough viruses with MN-rgp120 is an effective way complement and significantly expand antigenic complexity and increase breadth of cross neutralizing activity.

Abstract

Oligonucleotide sequences encoding gp120 polypeptides from breakthrough isolates of vaccine trials using MN-rgp120 and the encoded gp120 polypeptides are provided. Use of the gp120 polypeptides from one or more of the isolates in a subunit vaccine, usually together with MN-rgp120, can provide protection against HIV strains that are sufficiently different from the vaccine strain (e.g.; MN-rgp120) that the vaccine does not confer protection against those strains. Antibodies induced by the polypeptides are also provided.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application No. 60/676,737 (formerly application Ser. No. 08/676,737, filed Jul. 8, 1996, entitled "HIV Envelope Polypeptides and Vaccine" and naming Phillip W. Berman as the inventor.
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to HIV envelope polypeptides and vaccines containing the polypeptides.
2. Description of the Related Art
Acquired immunodeficiency syndrome (AIDS) is caused by a retrovirus identified as the human immunodeficiency virus (HIV). There have been intense efforts to develop a vaccine that induces a protective immune response based on induction of antibodies or cellular responses. Recent efforts have used subunit vaccines where an HIV protein, rather than attenuated or killed virus, is used as the immunogen in the vaccine for safety reasons. Subunit vaccines generally include gp120, the portion of the HIV envelope protein which is on the surface of the virus.
The HIV envelope protein has been extensively described, and the amino acid and nucleic acid sequences encoding HIV envelope from a number of HIV strains are known (Myers, G. et al., 1992. Human Retroviruses and AIDS. A compilation and analysis of nucleic acid and amino acid sequences. Los Alamos National Laboratory, Los Alamos, N. Mex.). The HIV envelope protein is a glycoprotein of about 160 kd (gp160) which is anchored in the membrane bilayer at its carboxyl terminal region. The N-terminal segment, gp120, protrudes into the aqueous environment surrounding the virion and the C-terminal segment, gp41, spans the membrane. Via a host-cell mediated process, gp160 is cleaved to form gp120 and the integral membrane protein gp41. As there is no covalent attachment between gp120 and gp41, free gp120 is sometimes released from the surface of virions and infected cells.
The gp120 molecule consists of a polypeptide core of 60,000 daltons which is extensively modified by N-linked glycosylation to increase the apparent molecular weight of the molecule to 120,000 daltons. The amino acid sequence of gp120 contains five relatively conserved domains interspersed with five hypervariable domains. The positions of the 18 cysteine residues in the gp120 primary sequence, and the positions of 13 of the approximately 24 N-linked glycosylation sites in the gp120 sequence are common to all gp120 sequences. The hypervariable domains contain extensive amino acid substitutions, insertions and deletions. Sequence variations in these domains result in up to 30% overall sequence variability between gp120 molecules from the various viral isolates. Despite this variation, all gp120 sequences preserve the ability of the virus to bind to the viral receptor CD4 and to interact with gp41 to induce fusion of the viral and host cell membranes.
gp120 has been the object of intensive investigation as a vaccine candidate for subunit vaccines, as the viral protein which is most likely to be accessible to immune attack. At present, clinical trials using gp120 MN strain are underway. However, to date no human vaccine trial has been of sufficient size to confirm or refute vaccine efficacy.
The development of candidate HIV-1 vaccines is burdened by the lack of in vivo or in vitro models of HIV-1 infection that accurately approximate the conditions of natural infection in humans. Several candidate HIV-1 vaccines [Berman et al.; J. Virol. 7:4464-9 (1992); Haigwood et al.; J. Virol. 66:172-82 (1992); Salmon-Ceron et al.; AIDS Res. and Human Retroviruses 11:1479-86 (1995)] have been described that elicit broadly cross-reactive antibodies able to neutralize a variety of diverse HIV-1 isolates in vitro. However, the relevance of in vitro assays to protective immunity in vivo is uncertain. Although several vaccines have provided chimpanzees with protection from challenge by homologous and heterologous strains of HIV-1, protection has not always correlated with in vitro neutralization assays carried out in T cell lines, or in lectin- and cytokine-activated peripheral blood mononuclear cells (PBMCs) [Berman et al.; Nature 345:622-5 (1990); Bruck et al.; Vaccine 12(12):1141-8 (1994); El-Amad et al.; AIDS 9:1313-22 (1995); Girard et al.; J. Virol. 69:6239-48 (1995); and Fulz et al; Science 256:1687-1690 (1992)]. While successful protection of chimpanzees is encouraging and has historically proved to be a reliable indicator of vaccine efficacy, the conditions of infection in all experimental models of HIV-1 infection differ significantly from natural infection in humans.
Experimental HIV-1 infection in vivo and in vitro both suffer from the limitation that the in vitro amplification of HIV-1, which is required to prepare virus stocks for in vitro or in vivo infectivity experiments, imposes a genetic selection that results in a spectrum of virus quasi-species that differ from the spectrum of variants present in the clinical specimens used to establish the culture [Kusumi et al.; J. Virol. 66:875 (1992); Meyerhans et al.; Cell 58:901-10 (1989)]. Because of these uncertainties, and even greater uncertainties related to the amount of virus transmitted, the site and cell type involved in initial replication, and the kinetics of virus dissemination, the ability of currently available in vitro or in vivo assays to reliably predict vaccine efficacy is questionable.
One of the candidate HIV-1 vaccines that have entered human clinical trials is recombinant gp120 prepared in Chinese hamster ovary (CHO) cells from the MN strain of HIV-1 (MN-rgp120) (Berman et al.; J. Virol. 7:4464-9 (1992)). To date, approximately 499 adults have participated in Phase 1 and 2 immunogenicity and safety trials of this vaccine. The data collected thus far suggest that MN-rgp120 is safe, immunogenic, and elicits high titers of neutralizing antibodies in greater than 95% of individuals immunized according to a 0, 1, and 6 month immunization schedule [Belshe et al.; JAMA 272(6):475-80 (1994); McElrath; Seminars in Cancer Biol. 6:1-11 (1995)]. However, during the course of these trials, nine vaccinees who received MN-rgp120 have become infected with HIV-1 through high risk behavior. Small trials, such as these, in populations with low rates of infection and minimally sized placebo control groups do not have sufficient statistical power to confirm or refute vaccine efficacy.
However, effective vaccines based on gp120 or another HIV protein for protection against additional strains of HIV are still being sought to prevent the spread of this disease.
DESCRIPTION OF THE BACKGROUND ART
Recombinant subunit vaccines are described in Berman et al., PCT/US91/02250 (published as number WO91/15238 on Oct. 17, 1991). See also, e.g. Hu et al., Nature 328:721-724 (1987) (vaccinia virus-HIV envelope recombinant vaccine); Arthur et al., J. Virol. 63(12): 5046-5053 (1989) (purified gp120); and Berman et al., Proc. Natl. Acad. Sci. U.S.A. 85:5200-5204 (1988) (recombinant envelope glycoprotein gp120).
Numerous sequences for gp120 are known. The sequence of gp120 from the IIIB substrain of HIV-1LAI referred to herein is that determined by Muesing et al., "Nucleic acid structure and expression of the human AIDS/lymphadenopathy retrovirus, Nature 313:450-458 (1985). The sequences of gp120 from the NY-5, Jrcsf, Z6, Z321, and HXB2 strains of HIV-1 are listed by Myers et al., "Human Retroviruses and AIDS; A compilation and analysis of nucleic acid and amino acid sequences," Los Alamos National Laboratory, Los Alamos, N. Mex. (1992). The sequence of the Thai isolate A244 is provided by McCutchan et al., "Genetic Variants of HIV-1 in Thailand," AIDS Res. and Human Retroviruses 8:1887-1895 (1992). The MN1984 clone is described by Gurgo et al., "Envelope sequences of two new United States HIV-1 isolates," Virol. 164: 531-536 (1988). As used herein, MN, MN-rgp120, the MN clone or isolate refers to MNGNE. The MNGNE amino acid sequence is Sequence ID NO:41.
Each of the above-described references is incorporated herein by reference in its entirety.
SUMMARY OF THE INVENTION
Oligonucleotide sequences encoding gp120 polypeptides from breakthrough isolates of vaccine trials using MN-rgp120 and the encoded gp120 polypeptides are provided. Use of the gp120 polypeptides from one or more of the isolates in a subunit vaccine, usually together with MN-rgp120, can provide protection against HIV strains that are sufficiently different from the vaccine strain (e.g.; MN-rgp120) that the vaccine does not confer protection against those strains. Antibodies induced by the polypeptides are also provided.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-1G illustrate the kinetics of antibody response to MN-rgp120 in vaccinees infected with HIV-1. Sera were collected at the time points indicated and assayed for antibodies reactive with MN-rgp120 (open circles) or a synthetic peptide derived from the V3 domain of MN-rgp120 (closed circles). Arrows indicate dates of injection. Plus sign indicates the first time HIV-1 infection was detected. Shaded area indicates data collected after HIV-1 infection. Data from vaccinee C6 is shown in FIG. 1A; C8 in FIG. 1B; C7, FIG. 1C; C11, FIG. 1D; C10, FIG. 1E; C17, FIG. 1F; and C15, FIG. 1G.
FIGS. 2A-2G illustrate the kinetics of CD4 blocking antibody response in vaccinees infected with HIV-1. Sera were collected at the time points indicated and assayed for antibodies able to block the binding of [125 I]-labeled MN-rgp120 to cell surface CD4. Arrows indicate dates of injection. Plus sign indicates the first time HIV-1 infection was detected. Shaded area indicates data collected after HIV-1 infection. Data from vaccinee C6 is shown in FIG. 2A; C8 in FIG. 2B; C7, FIG. 2C; C11, FIG. 2D; C10, FIG. 2E; C17, FIG. 2F; and C15, FIG. 2G.
FIGS. 3A-3J illustrate predicted amino acid sequences of envelope glycoproteins (gp120) from breakthrough viruses. Proviral DNA sequences were amplified by PCR from PBMCs and cloned into the PRK5 expression plasmid. Two clones from each infected vaccinee were sequenced from double stranded plasmid DNA. Sequence numbering is with reference to the initiator methionine residue of gp120. For the purpose of comparison, the sequences shown begin at amino acid 12 of the mature, fully processed, envelope glycoproteins (corresponding to position 41 of the gp120 open reading frame). Shaded areas indicate sequences at neutralizing epitopes, dark boxes indicate polymorphisms thought to be important for the binding of virus neutralizing MAbs reactive with MN-rgp120. Conserved (C) regions and variable (V) regions are indicated above the sequences. Boxes indicate sequence homologies and polymorphisms.
FIG. 4 illustrates immunoprecipitation of recombinant gp120 prepared from breakthrough viruses. Recombinant gp120s from the seven breakthrough viruses were prepared by transient transfection of 293s cells. Cells were metabolically labeled with 35 S methionine and growth conditioned cell culture supernatants were immunoprecipitated with polyclonal antisera to MN-rgp120. Immunoprecipitates were resolved by SDS-PAGE and visualized by autoradiography. C8 lanes a and b correspond to clones C8.3 and C8.6; C6 lanes a and b correspond to clones C6.1 and C6.5; C7 lanes a and b correspond to clones C7.2 and C7.10; C17 lanes a and b correspond to C17.1 and C17.3; C11 lanes a and b correspond to clones C11.5 and C11.7; C10 lanes a and b correspond to clones C10.5 and C10.7; C15 lanes a and b correspond to clones C15.2 and C15.3.
FIGS. 5A-5D illustrate binding of monoclonal antibodies to recombinant gp120 from breakthrough viruses. Growth-conditioned cell culture supernatants were collected from 293s cells transiently transfected with plasmids directing the expression of breakthrough virus envelope glycoproteins. The relative rgp120 concentrations were determined by ELISA using MAb 5B6 specific for the HSV-1 glycoprotein D flag epitope at the amino terminus of all of the rgp120 variants described herein. The resulting rgp120 preparations were captured onto wells of microtiter plates coated with a polyclonal antibody specific for a conserved sequence in the C-terminus of gp120. The binding of virus neutralizing monoclonal antibodies reactive with gp120 was determined by ELISA. FIG. 5A, binding by MAb (5B6) specific for the HSV-1 glycoprotein D flag epitope; FIG. 5B, binding by MAb (1034) against the V3 domain of MN-rgp120; FIG. 5C binding by MAb (50.1) raised against a synthetic peptide corresponding to the V3 domain of MN-rgp120; FIG. 5D, binding by a human MAb (15e) known to block the binding of gp120 to CD4.
FIG. 6 depicts the mature envelope glycoprotein (gp120) from the MN clone of the MN strain of HIV-1 (SEQ. ID. NO:41). Hypervariable domains are indicated in bold, and the V and C regions are indicated (according to Modrow et al., J. Virology 61(2) :570 (1987). Potential glycosylation sites are marked with a (*).
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides gp120 polypeptides from breakthrough isolates of HIV vaccine trials. Novel oligonucleotide sequences encoding gp120 from breakthrough isolates which can be used to express gp120 are also provided. Use of gp120 polypeptides from one or more of the isolates in a subunit vaccine, usually together with MN-rgp120, can provide protection against HIV strains that are sufficiently different from the vaccine strain (e.g.; MN-rgp120) that the vaccine does not confer protection against those strains.
In one embodiment, the vaccine is based on the use of the MN-rgp120 polypeptide (Sequence ID No:41) and gp120 polypeptides from MN-like viruses that include neutralizing epitopes that are not present in the initial vaccine strain, and are sufficiently different from those of the vaccine strain, to have been able to cause HIV-1 infections in MN-rgp120 vaccinated individuals (i.e.; to result in breakthrough infections). Use of the initial vaccine strain empirically determines the viruses present in the population that contain additional neutralizing epitopes sufficiently different from those of the vaccine strain to escape protection induced by the vaccine strain. Use of an initial representative gp120 polypeptide in a vaccine acts as a sieve so that viruses that are not effectively protected against by the vaccine strain breakthrough the vaccine, empirically resulting in determination of additional strains in a given geographic region that are not protected against by the initial vaccine strain. Use of gp120 from those breakthrough isolates complements the vaccine isolate by providing additional neutralizing epitopes not present in the initial vaccine strain, therefore creating a more complete vaccine that confers protection against multiple different virus strains in the region.
Prior HIV-1 vaccine strategies were based on selection of appropriate candidate vaccine polypeptides based on homology alignment studies. However, since some of the neutralizing epitopes are conformation-dependent and the location of all of these epitopes is not known, this approach necessarily cannot determine all of the neutralizing epitopes that should be included in a vaccine for a particular region. In contrast, the present approach uses a selected representative strain and empirically determines strains that are sufficiently different and therefore breakthrough the barrier of protection provided by the initial vaccination program. Those strains can be included in the vaccine to confer more complete protection from HIV strains in the region. In addition, those strains can be used alone to confer protection against the breakthrough virus.
In another embodiment, the invention comprises a vaccine containing a first HIV gp120 polypeptide sequence and a breakthrough isolate HIV gp120 polypeptide sequence from a vaccinee vaccinated with a vaccine including the first HIV gp120 polypeptide sequence, the HIV gp120 polypeptide sequences being in a suitable carrier. Fragments of one or both HIV gp120 polypeptide sequences can be substituted for one or both of the corresponding HIV gp120 polypeptide sequences.
Preferably, the first gp120 polypeptide sequence contains neutralizing epitopes found in one or more gp120 polypeptides present in isolates from the geographical region where the initial vaccine (i.e., the vaccine that gives rise to the breakthrough isolate) is administered. More preferably, the first gp120 polypeptide sequence contains at least one of the more common neutralizing epitopes for the region, and most preferably the first gp120 polypeptide sequence contains at least one of the three most common neutralizing epitopes.
gp120 polypeptide sequences suitable for use as the first gp120 polypeptide sequence include gp120 MN, the Thai isolate A244 sequence (hereinafter "gp120 A244"), gp120 MN-GNE6 (Sequences ID NOs:43 and 44; also known in the art as "gp120 GNE6"), and gp120 MN-GNE8 (Sequence ID NO:46; also known in the art as "gp120 GNE8"), and the like. gp120 MN, gp120 MN-GNE6, and gp120 MN-GNE8 are especially preferred for use as the first gp120 polypeptide sequence in initial vaccines for North America. gp120 A244 is especially preferred for use as the first gp120 polypeptide sequence in initial vaccines for Thailand.
In a variation of this embodiment, the vaccine includes two different (i.e., first and second) gp120 polypeptide sequences, or fragments thereof, in combination with a breakthrough isolate HIV gp120 polypeptide sequence. The latter can be from a vaccinee vaccinated with either or both of the first and second HIV gp120 polypeptide sequences.
Exemplary vaccines include those containing combinations of gp120 MN, gp120 A244, gp120 MN-GNE6 (Sequence ID NOs:43 and 44), and gp120 MN-GNE8 (Sequence ID NO:46). Combinations of gp120 MN and gp120 A244 or gp120 MN-GNE8 (Sequence ID NO:46) with a breakthrough isolate HIV gp120 polypeptide sequence are especially preferred.
In vaccines containing gp120 MN, the breakthrough isolate HIV gp120 polypeptide sequence can be an HIV gp120 polypeptide sequence selected from the group consisting of Sequence ID NOs:2, 5, 8, 10, 12, 16, 19, 23, 25, 28, 31, 33, 36, and 39, and fragments thereof.
The term "subunit vaccine" is used herein, as in the art, to refer to a viral vaccine that does not contain virus, but rather contains one or more viral proteins or fragments of viral proteins. As used herein, the term "multivalent", means that the vaccine contains gp120 from at least two HIV isolates having different amino acid sequences.
The term "breakthrough isolate" or "breakthrough virus" is used herein, as in the art, to refer to a virus isolated from a vaccinee.
The terms "amino acid sequence", "polypeptide sequence", and "polypeptide" are used interchangeably herein as in the art, as are the terms "nucleic acid sequence", "nucleotide sequence", and "oligonucleotide".
Polypeptides from Breakthrough Isolates
The gp120 polypeptides of this invention correspond to the amino acid sequences of seven breakthrough isolates which are illustrated below in Table 1. A polypeptide of this invention includes an HIV gp120 amino acid sequence illustrated in Table 1 (Sequence ID NOs:1, 4, 7, 9, 11, 15, 18, 22, 24, 27, 30, 32, 35, and 38) and fragments thereof. The polypeptides of this invention can include fused sequences from two or more HIV gp120 or gp160 amino acid sequences.
The polypeptide can also be joined to another viral protein, such as a flag epitope amino acid sequence. The term "flag epitope" is used herein, as in the art, to denote an amino acid sequence that includes an epitope recognized by a monoclonal antibody. Flag epitopes facilitate using single monoclonal antibody affinity purification of a plurality of different recombinant proteins, each having the flag epitope recognized by the monoclonal antibody. Numerous amino acid sequences can function as flag epitopes. The N-terminal sequences of Herpes Simplex Virus Type 1 (HSV-1) glycoprotein D (gD-1) is conveniently used as the flag epitope and its use is described in detail in the examples. The flag epitope is conveniently fused to the N terminus of the HIV gp120 polypeptide sequence. Alternatively, however, monoclonal antibodies that recognize neutralizing epitopes in the rgp120 sequences can be used to affinity purify the amino acid sequences, and a flag epitope can be omitted.
In addition, various signal sequences can be joined to a polypeptide of this invention. Although rgp120 is secreted to some extent in HIV cultures, the amount of the envelope glycoprotein released from (secreted by) the host cells varies widely from strain to strain. Various signal sequences can be introduced into the polypeptide by joining a nucleotide sequence encoding the signal sequence to the nucleotide sequence encoding the rgp120 to facilitate secretion of rgp120 from the cells. For example, Chiron HIV gp120 polypeptides include a signal sequence from tissue plasminogen activator (TPA) that provides good secretion of rgp120. Additional signal sequences are well known and include the N-terminal domain of murine leukemia virus surface protein gp70 described by Kayman et al., J. Virol. 68:400-410 (1984).
Table 1 illustrates the nucleotide and deduced amino acid sequences for two clones of each the seven breakthrough isolates of this invention. The clones are: C6.1; C6.5; C8.3; C8.6; C15.2; C15.3; C7.2; C7.10; C11.5; C11.7; C10.5; C10.7; C17.1; and C17.3. These sequences are SEQ. ID. NOs:1-40. The amino acid sequence for MN and the nucleotide and deduced amino acid sequences for MN-GNE6 and MN-GNE8 are illustrated in the sequence listing hereinafter. In the listing for MN-GNE6, a stop codon appears at amino acid residue position 51. This stop codon can be replaced with a codon encoding the corresponding amino acid from MN or MN-GNE8 or another isolate.
                                  TABLE 1                                 
__________________________________________________________________________
CLONE C6.1                                                                
     GGG GTA CCT GTG TGG AAG GAA GCA ACC ACC ACT CTA                      
                                36                                        
     Gly Val Pro Val Trp Lys Glu Ala Thr Thr Thr Leu                      
      1               5                  10                               
TTT TGT GCA TCA GAT GCT AAA GCA TAT GAC ACA GAG GTG                       
                                75                                        
Phe Cys Ala Ser Asp Ala Lys Ala Tyr Asp Thr Glu Val                       
          15                  20                  25                      
CAT AAT GTT TGG GCC ACA CAT GCT TGT GTA CCC ACA GAC                       
                                114                                       
His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp                       
                 30                  35                                   
CCA AAC CCA CAA GAA ATG GTA TTG GAA AAT GTG ACA GAA                       
                                153                                       
Pro Asn Pro Gln Glu Met Val Leu Glu Asn Val Thr Glu                       
     40                  45                  50                           
GAT TTT AAC ATG TGG AAA AAT GAC ATG GTA GAA CAG ATG                       
                                192                                       
Asp Phe Asn Met Trp Lys Asn Asp Met Val Glu Gln Met                       
             55                  60                                       
CAT GAG GAT ATA ATC AGT TTA TGG GAT CAA AGC CTA AAA                       
                                231                                       
His Glu Asp Ile Ile Ser Leu Trp Asp Gln Ser Leu Lys                       
 65                  70                  75                               
CCA TGT GTA AAA TTA ACC CCA CTC TGT ATT ACT TTA AAT                       
                                270                                       
Pro Cys Val Lys Leu Thr Pro Leu Cys Ile Thr Leu Asn                       
         80                  85                  90                       
TGC ACC AAT TGG AAG AAG AAT GAT ACT AAA ACT AAT AGT                       
                                309                                       
Cys Thr Asn Trp Lys Lys Asn Asp Thr Lys Thr Asn Ser                       
                 95                 100                                   
AGT AGT ACT ACA ACT AAT AAT AGT AGT GCT ACA GCT AAT                       
                                348                                       
Ser Ser Thr Thr Thr Asn Asn Ser Ser Ala Thr Ala Asn                       
    105                 110                 115                           
AGT AGT AGT ACT ACA ACT AAT AGT AGT TGG GGA GAG ATA                       
                                387                                       
Ser Ser Ser Thr Thr Thr Asn Ser Ser Trp Gly Glu Ile                       
            120                 125                                       
AAG GAG GGA GAA ATA AAG AAC TGC TCT TTC AAT ATC ACC                       
                                426                                       
Lys Glu Gly Glu Ile Lys Asn Cys Ser Phe Asn Ile Thr                       
130                 135                 140                               
ACA AGC ATA AGA GAC AAG GTG AAG AAA GAA TAT GCA CTT                       
                                465                                       
Thr Ser Ile Arg Asp Lys Val Lys Lys Glu Tyr Ala Leu                       
        145                 150                 155                       
TTT TAT AGC CTT GAT GTA GTA CCA ATA GAA AAT GAT AAT                       
                                504                                       
Phe Tyr Ser Leu Asp Val Val Pro Ile Glu Asn Asp Asn                       
                160                 165                                   
ACT AGC TAT AGG TTG AGA AGT TGT AAC ACC TCA GTC ATT                       
                                543                                       
Thr Ser Tyr Arg Leu Arg Ser Cys Asn Thr Ser Val Ile                       
    170                 175                 180                           
ACA CAA GCC TGT CCA AAG GTA ACT TTT GAG CCA ATT CCC                       
                                582                                       
Thr Gln Ala Cys Pro Lys Val Thr Phe Glu Pro Ile Pro                       
            185                 190                                       
ATA CAT TAT TGT ACC CCG GCT GGT TTT GCG ATT CTG AAG                       
                                621                                       
Ile His Tyr Cys Thr Pro Ala Gly Phe Ala Ile Leu Lys                       
195                 200                 205                               
TGT AGA GAT AAA AAG TTC AAT GGA ACA GGA CCA TGC AAA                       
                                660                                       
Cys Arg Asp Lys Lys Phe Asn Gly Thr Gly Pro Cys Lys                       
        210                 215                 220                       
AAT GTT AGC ACA GTA CAA TGT GCA CAT GGA ATT AAG CCA                       
                                699                                       
Asn Val Ser Thr Val Gln Cys Ala His Gly Ile Lys Pro                       
                225                 230                                   
GTA GTG TCA ACT CAA CTG CTG TTA AAT GGC AGC CTA GCA                       
                                738                                       
Val Val Ser Thr Gln Leu Leu Leu Asn Gly Ser Leu Ala                       
    235                 240                 245                           
GAA GAA GAG GTA ATA ATT AGA TCT GCC AAT TTC TCA AAC                       
                                777                                       
Glu Glu Glu Val Ile Ile Arg Ser Ala Asn Phe Ser Asn                       
            250                 255                                       
AAT GCT AAA ATC ATA ATA GTA CAG TTG AGG GAA CCT GTA                       
                                816                                       
Asn Ala Lys Ile Ile Ile Val Gln Leu Arg Glu Pro Val                       
260                 265                 270                               
GAA ATT AAT TGT ACA AGA CCC AGC AAC AAT ACA ATA AAA                       
                                855                                       
Glu Ile Asn Cys Thr Arg Pro Ser Asn Asn Thr Ile Lys                       
        275                 280                 285                       
GGT ATA CAC ATA GGA CCA GGG AGA GCA TTT TAT GCA ACA                       
                                894                                       
Gly Ile His Ile Gly Pro Gly Arg Ala Phe Tyr Ala Thr                       
                290                 295                                   
GGA GAC ATA CGA GGA GAT ATA AGA CAA GCA CAT TGT AAC                       
                                933                                       
Gly Asp Ile Arg Gly Asp Ile Arg Gln Ala His Cys Asn                       
    300                 305                 310                           
ATT AGT GGA GCA AAA TGG AAT AAC ACT TTA AAG AAG GTA                       
                                972                                       
Ile Ser Gly Ala Lys Trp Asn Asn Thr Leu Lys Lys Val                       
            315                 320                                       
GTT AAA AAA TTA AAA GAA CAA TTT CCA AAT AAA ACA ATA                       
                                1011                                      
 Val Lys Lys Leu Lys Glu Gln Phe Pro Asn Lys Thr Ile                      
 325                 330                 335                              
GTC TTT AAC CAT TCC TCA GGA GGG GAC CCA GAA ATT GTA                       
                                1050                                      
Val Phe Asn His Ser Ser Gly Gly Asp Pro Glu Ile Val                       
        340                 345                 350                       
ATG CAC AGT TTT AAT TGT CAA GGG GAA TTT TTC TAC TGT                       
                                1089                                      
Met His Ser Phe Asn Cys Gln Gly Glu Phe Phe Tyr Cys                       
                355                 360                                   
AAT ACA ACA AAG CTG TTT AAT AGT ACT TGG AAT GAT ACT                       
                                1128                                      
Asn Thr Thr Lys Leu Phe Asn Ser Thr Trp Asn Asp Thr                       
    365                 370                 375                           
ACA GAG TCA AAT AAC AAT GAT AGT ACT ATT ACA CTC CCA                       
                                1167                                      
Thr Glu Ser Asn Asn Asn Asp Ser Thr Ile Thr Leu Pro                       
            380                 385                                       
TGC AGA ATA AAA CAA ATT ATA AAC ATG TGG CAG GAA ATA                       
                                1206                                      
Cys Arg Ile Lys Gln Ile Ile Asn Met Trp Gln Glu Ile                       
390                 395                 400                               
GGA AAA GCA ATG TAT GCC CCT CCC ACC AGA GGA GAA ATT                       
                                1245                                      
Gly Lys Ala Met Tyr Ala Pro Pro Thr Arg Gly Glu Ile                       
        405                 410                 415                       
AAA TGT TCA TCA AAT ATT ACA GGA CTA CTG TTA ATA AGA                       
                                1284                                      
Lys Cys Ser Ser Asn Ile Thr Gly Leu Leu Leu Ile Arg                       
                420                 425                                   
GAT GGT GGT ATT AAC ACT AGC GAT GCC ACC GAG ACC TTC                       
                                1323                                      
Asp Gly Gly Ile Asn Thr Ser Asp Ala Thr Glu Thr Phe                       
    430                 435                 440                           
AGA CCG GGA GGA GGA GAT ATG AGG GAC AAT TGG AGA AGT                       
                                1362                                      
Arg Pro Gly Gly Gly Asp Met Arg Asp Asn Trp Arg Ser                       
            445                 450                                       
GAA TTA TAT AAA TAT AAA GTA GTG AAA ATT GAG CCA TTA                       
                                1401                                      
Glu Leu Tyr Lys Tyr Lys Val Val Lys Ile Glu Pro Leu                       
455                 460                 465                               
GGA GTA GCA CCC ACC AAG GCA AAG AGA AGA GTG GTG CAG                       
                                1440                                      
Gly Val Ala Pro Thr Lys Ala Lys Arg Arg Val Val Gln                       
        470                 475                 480                       
AGA GAA AAA AGA GCA GTA ACA CTA GGA GCT ATG TTC CTT                       
                                1479                                      
Arg Glu Lys Arg Ala Val Thr Leu Gly Ala Met Phe Leu                       
                485                 490                                   
GGG TTC TTA GGA GCA TAA AGC TTC 1503                                      
Gly Phe Leu Gly Ala Xaa Ser Phe                                           
    495                 500 501                                           
CLONE C6.5                                                                
    GGG GTA CCT GTA TGG AAA GAA GCA ACC ACC ACT CTA                       
                                36                                        
    Gly Val Pro Val Trp Lys Glu Ala Thr Thr Thr Leu                       
       1               5                  10                              
TTT TGT GCA TCA GAT GCT AAA GCA TAT GAC ACA GAG GTG                       
                                75                                        
Phe Cys Ala Ser Asp Ala Lys Ala Tyr Asp Thr Glu Val                       
         15                  20                  25                       
CAT AAT GTT TGG GCC ACA CAT GCT TGT GTA CCC ACA GAC                       
                                114                                       
His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp                       
                 30                  35                                   
CCA AAC CCA CAA GAA ATG GTA TTG GAA AAT GTG ACA GAA                       
                                153                                       
Pro Asn Pro Gln Glu Met Val Leu Glu Asn Val Thr Glu                       
     40                  45                  50                           
GAT TTT AAC ATG TGG AAA AAT GAC ATG GTA GAA CAG ATG                       
                                192                                       
Asp Phe Asn Met Trp Lys Asn Asp Met Val Glu Gln Met                       
             55                  60                                       
CAT GAG ANT ATA ATC AGT TTA TGG GAT CAA AGC CTA AAA                       
                                231                                       
His Glu Xaa Ile Ile Ser Leu Trp Asp Gln Ser Leu Lys                       
 65                  70                  75                               
CCA TGT GTA AAA TTA ACC CCA CTC TGT ATT ACT TTA AAT                       
                                270                                       
Pro Cys Val Lys Leu Thr Pro Leu Cys Ile Thr Leu Asn                       
        80                  85                  90                        
TGC ACC AAT TGG AAG GAG AAT GAT ACT AAA ACT AAT AGT                       
                                309                                       
Cys Thr Asn Trp Lys Glu Asn Asp Thr Lys Thr Asn Ser                       
                 95                 100                                   
AGT AGT ACT ACA ACT AAT AAT AGT AGT GCT ACA GCT AAT                       
                                348                                       
Ser Ser Thr Thr Thr Asn Asn Ser Ser Ala Thr Ala Asn                       
    105                 110                 115                           
AGT AGT AGT ACT ACA ACT AAT AGT AGT TGG GGA GAG ATA                       
                                387                                       
Ser Ser Ser Thr Thr Thr Asn Ser Ser Trp Gly Glu Ile                       
            120                 125                                       
AAG GAG GGA GAA ATA AAG AAC TGC TCT TTC AAT ATC ACC                       
                                426                                       
Lys Glu Gly Glu Ile Lys Asn Cys Ser Phe Asn Ile Thr                       
130                 135                 140                               
ACA GGC ATA AGA GAC AAG GTG AAG AAA GAA TAT GCA CTT                       
                                465                                       
Thr Gly Ile Arg Asp Lys Val Lys Lys Glu Tyr Ala Leu                       
        145                 150                 155                       
TTT TAT AGC CTT GAT GTA GTA CCA ATA GAA AAT GAT AAT                       
                                504                                       
Phe Tyr Ser Leu Asp Val Val Pro Ile Glu Asn Asp Asn                       
                160                 165                                   
ACT AGC TAT AGG TTG AGA AGT TGT AAC ACC TCA GTC ATT                       
                                543                                       
Thr Ser Tyr Arg Leu Arg Ser Cys Asn Thr Ser Val Ile                       
    170                 175                 180                           
ACA CAA GCC TGT CCA AAG GTA ACT TTT GAG CCA ATT CCC                       
                                582                                       
Thr Gln Ala Cys Pro Lys Val Thr Phe Glu Pro Ile Pro                       
            185                 190                                       
ATA CAT TAT TGT ACC CCG GCT GGT TTT GCG ATT CTG AAG                       
                                621                                       
Ile His Tyr Cys Thr Pro Ala Gly Phe Ala Ile Leu Lys                       
195                 200                 205                               
TGT AAA GAT AAA AAG TTC AAT GGA ACA GGA CCA TGC AAA                       
                                 660                                      
Cys Lys Asp Lys Lys Phe Asn Gly Thr Gly Pro Cys Lys                       
        210                 215                 220                       
AAT GTT AGC ACA GTA CAA TGT ACA CAT GGA ATT AAG CCA                       
                                 699                                      
Asn Val Ser Thr Val Gln Cys Thr His Gly Ile Lys Pro                       
                225                 230                                   
GTA GTG TCA ACT CAA CTG CTG TTA AAT GGC AGC CTA GCA                       
                                 738                                      
Val Val Ser Thr Gln Leu Leu Leu Asn Gly Ser Leu Ala                       
    235                 240                 245                           
GAA GAA GAG GTA ATA ATT AGA TCT GCC AAT TTC TCA AAC                       
                                777                                       
Glu Glu Glu Val Ile Ile Arg Ser Ala Asn Phe Ser Asn                       
            250                 255                                       
AAT GCT AAA ATC ATA ATA GTA CAG TTG AAG GAA CCT GTA                       
                                 816                                      
Asn Ala Lys Ile Ile Ile Val Gln Leu Lys Glu Pro Val                       
260                 265                 27                                
GAA ATT AAT TGT ACA AGA CCC AGC AAC AAT ACA ATA AAA                       
                                 855                                      
Glu Ile Asn Cys Thr Arg Pro Ser Asn Asn Thr Ile Lys                       
        275                 280                 285                       
GGT ATA CAC ATA GGA CCA GGG AGA GCA TTT TAT GCA ACA                       
                                 894                                      
Gly Ile His Ile Gly Pro Gly Arg Ala Phe Tyr Ala Thr                       
                290                 295                                   
GGA GAC ATA CGA GGA GAT ATA AGA CAA GCA CAT TGT AAC                       
                                933                                       
Gly Asp Ile Arg Gly Asp Ile Arg Gln Ala His Cys Asn                       
    300                 305                 310                           
ATT AGT GGA GCA AAA TGG AAT AAC ACT TTA AAG AAG GTA                       
                                972                                       
Ile Ser Gly Ala Lys Trp Asn Asn Thr Leu Lys Lys Val                       
            315                 320                                       
GTT ATA AAA TTA AAA GAA CAA TTT CCA AAT AAA ACA ATA                       
                                1011                                      
Val Ile Lys Leu Lys Glu Gln Phe Pro Asn Lys Thr Ile                       
325                 330                 335                               
GTC TTT AAC CAT TCC TCA GGA GGG GAC CCA GAA ATT GTA                       
                                1050                                      
Val Phe Asn His Ser Ser Gly Gly Asp Pro Glu Ile Val                       
        340                 345                 350                       
ATG CAC AGT TTT AAT TGT CAA GGG GAA TTT TTC TAC TGT                       
                                1089                                      
Met His Ser Phe Asn Cys Gln Gly Glu Phe Phe Tyr Cys                       
                355                 360                                   
AAT ACA ACG AAG CTG TTT AAT AGT ACT TGG AAT GAT ACT                       
                                1128                                      
Asn Thr Thr Lys Leu Phe Asn Ser Thr Trp Asn Asp Thr                       
    365                 370                 375                           
ACA GAG TCA AAT AAC AAT GAT AGT ACT ATT ACA CTC CCA                       
                                1167                                      
Thr Glu Ser Asn Asn Asn Asp Ser Thr Ile Thr Leu Pro                       
            380                 385                                       
TGC AGA ATA AAA CAA ATT ATA AAC ATG TGG CAG GAA GTA                       
                                1206                                      
Cys Arg Ile Lys Gln Ile Ile Asn Met Trp Gln Glu Val                       
390                 395                 400                               
GGA AAA GCA ATG TAT GCC CCT CCC ATC AGA GGA GAA ATT                       
                                1245                                      
Gly Lys Ala Met Tyr Ala Pro Pro Ile Arg Gly Glu Ile                       
        405                 410                 415                       
AAA TGT TCA TCA AAT ATT ACA GGA CTA CTG TTA ACA AGA                       
                                1284                                      
Lys Cys Ser Ser Asn Ile Thr G1y Leu Leu Leu Thr Arg                       
                420                 425                                   
GAT GGT GGT ATT AAC ACT AGC GAT GCC ACC GAG ACC TTC                       
                                1323                                      
Asp Gly Gly Ile Asn Thr Ser Asp Ala Thr Glu Thr Phe                       
    430                 435                 440                           
AGA CCG GGA GGA GGA GAT ATG AGG GAC AAT TGG AGA AGT                       
                                1362                                      
Arg Pro Gly Gly Gly Asp Met Arg Asp Asn Trp Arg Ser                       
            445                 450                                       
GAA TTA TAT AAA TAT AAA GTA GTG AAA ATT GAG CCA TTA                       
                                1401                                      
Glu Leu Tyr Lys Tyr Lys Val Val Lys Ile Glu Pro Leu                       
455                 460                 465                               
GGA GTA GCA CCC ACC AAG GCA AAG AGA AGA GTG GTG CAG                       
                                1440                                      
Gly Val Ala Pro Thr Lys Ala Lys Arg Arg Val Val Gln                       
        470                 475                 480                       
AGA GAA AAA AGA GCA GTA ACA CTA GGA GCT ATG TTC CTT                       
                                1479                                      
Arg Glu Lys Arg Ala Val Thr Leu Gly Ala Met Phe Leu                       
                485                 490                                   
GGG TTC TTG GGA GCA TAA AGC TTC 1503                                      
Gly Phe Leu Gly Ala Xaa Ser Phe                                           
    495                 500 501                                           
CLONE C8.3                                                                
G   GTA CCT GTA TGG AAA GAA GCA ACC ACC ACT CTA TTT                       
                                37                                        
    Val Pro Val Trp Lys Glu Ala Thr Thr Thr Leu Phe                       
      1               5                  10                               
TGT GCA TCA GAT GCT AAA GCA TAT GAT ACA GAG GTA CAT                       
                                76                                        
Cys Ala Ser Asp Ala Lys Ala Tyr Asp Thr Glu Val His                       
         15                  20                  25                       
AAT GTT TGG GCT ACA CAT GCC TGT GTA CCC ACA GAC CCC                       
                                115                                       
Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro                       
                 30                  35                                   
AAC CCA CAA GAA GTA GTA TTG GAA AAT GTA ACA GAA AAT                       
                                154                                       
Asn Pro Gln Glu Val Val Leu Glu Asn Val Thr Glu Asn                       
     40                  45                  50                           
TTT AAC ATG TGG AAA AAT AAC ATG GTA GAA CAG ATG CAT                       
                                193                                       
Phe Asn Met Trp Lys Asn Asn Met Val Glu Gln Met His                       
             55                  60                                       
GAG GAT ATA ATC AGT TTA TGG GAT CAA AGT CTA AAG CCA                       
                                232                                       
Glu Asp Ile Ile Ser Leu Trp Asp Gln Ser Leu Lys Pro                       
 65                  70                  75                               
TGT GTA AAA TTA ACC CCA CTC TGT GTT ACT TTA AAT TGC                       
                                271                                       
Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu Asn Cys                       
         80                  85                  90                       
ACT AAT TTG GAG AAT GCT AAT AAT ACC GAG AAT GCT AAT                       
                                310                                       
Thr Asn Leu Glu Asn Ala Asn Asn Thr Glu Asn Ala Asn                       
                 95                 100                                   
AAT ACC AAT AAT TAT ACC TTG GGG ATG GAG AGA GGT GAA                       
                                349                                       
Asn Thr Asn Asn Tyr Thr Leu Gly Met Glu Arg Gly Glu                       
    105                 110                 115                           
ATA AAA AAC TGC TCT TTC AAT ATC ACC ACA AGC TTA AGA                       
                                388                                       
Ile Lys Asn Cys Ser Phe Asn Ile Thr Thr Ser Leu Arg                       
            120                 125                                       
GAT AAG GTG AAA AAA GAA TAT GCA TTG TTT TAT AAA CTT                       
                                427                                       
Asp Lys Val Lys Lys Glu Tyr Ala Leu Phe Tyr Lys Leu                       
130                 135                 140                               
GAT GTA GTA CAA ATA GAT AAT AGT ACC AAC TAT AGG CTG                       
                                466                                       
Asp Val Val Gln Ile Asp Asn Ser Thr Asn Tyr Arg Leu                       
        145                 150                 155                       
ATA AGT TGT AAT ACC TCA GTC ATT ACA CAG GCC TGT CCA                       
                                505                                       
Ile Ser Cys Asn Thr Ser Val Ile Thr Gln Ala Cys Pro                       
                160                 165                                   
AAG GTA TCC TTT GAG CTA ATT CCC ATA CAT TAT TGT GCC                       
                                544                                       
Lys Val Ser Phe Glu Leu Ile Pro Ile His Tyr Cys Ala                       
    170                 175                 180                           
CCG GCT GGT TTT GCG ATT CTA AAG TGT AAA GAT AAG AAG                       
                                583                                       
Pro Ala Gly Phe Ala Ile Leu Lys Cys Lys Asp Lys Lys                       
            185                 190                                       
TTC AAT GGA ACA GGA CCA TGT AAA AAT GTC AGC ACA GTA                       
                                622                                       
Phe Asn Gly Thr Gly Pro Cys Lys Asn Val Ser Thr Val                       
195                 200                 205                               
CAA TGT ACA CAT GGA ATT AGA CCA GTA GTA TCA ACT CAA                       
                                661                                       
Gln Cys Thr His Gly Ile Arg Pro Val Val Ser Thr Gln                       
        210                 215                 220                       
CTA CTG TTA AAT GGC AGT CTA GCA GAA GAA GAG ATA GTA                       
                                700                                       
Leu Leu Leu Asn Gly Ser Leu Ala Glu Glu Glu Ile Val                       
                225                 230                                   
ATT AGA TCT GAA AAT ATC ACA GAC AAT GCT AAA ACC ATA                       
                                739                                       
Ile Arg Ser Glu Asn Ile Thr Asp Asn Ala Lys Thr Ile                       
    235                 240                 245                           
ATA GTG CAG CTA AAT GAA TCT ATA GTG ATT AAT TGT ACA                       
                                778                                       
Ile Val Gln Leu Asn Glu Ser Ile Val Ile Asn Cys Thr                       
            250                 255                                       
AGA CCC AAT AAC AAC ACA AGA AAA AGT ATA AAT ATA GGA                       
                                817                                       
Arg Pro Asn Asn Asn Thr Arg Lys Ser Ile Asn Ile Gly                       
260                 265                 270                               
CCA GGG AGA GCA TTC TAT ACA ACA GGA GAC ATA ATA GGA                       
                                856                                       
Pro Gly Arg Ala Phe Tyr Thr Thr Gly Asp Ile Ile Gly                       
        275                 280                 285                       
GAT ATA AGA CAA GCA CAT TGT AAC CTT AGT AAA ACA CAA                       
                                895                                       
Asp Ile Arg Gln Ala His Cys Asn Leu Ser Lys Thr Gln                       
                290                 295                                   
TGG GAA AAA ACG TTA AGA CAG ATA GCT ATA AAA TTA GAA                       
                                934                                       
Trp Glu Lys Thr Leu Arg Gln Ile Ala Ile Lys Leu Glu                       
    300                 305                 310                           
GAA AAA TTT AAG AAT AAA ACA ATA GCC TTT AAT AAA TCC                       
                                973                                       
Glu Lys Phe Lys Asn Lys Thr Ile Ala Phe Asn Lys Ser                       
            315                 320                                       
TCA GGA GGG GAC CCA GAA ATT GTA ATG CAC AGT TTT AAT                       
                                1012                                      
Ser Gly Gly Asp Pro Glu Ile Val Met His Ser Phe Asn                       
325                 330                 335                               
TGT GGA GGG GAA TTT TTC TAC TGT AAT ACA ACA AAA CTG                       
                                1051                                      
Cys Gly Gly Glu Phe Phe Tyr Cys Asn Thr Thr Lys Leu                       
        340                 345                 350                       
TTT AAT AGT ACC TGG AAT TTA ACA CAA CCG TTT AGT AAT                       
                                1090                                      
Phe Asn Ser Thr Trp Asn Leu Thr Gln Pro Phe Ser Asn                       
                355                 360                                   
ACC GGG AAT CGT ACT GAA GAG TTA AAT ATT ACA CTC CCA                       
                                1129                                      
Thr Gly Asn Arg Thr Glu Glu Leu Asn Ile Thr Leu Pro                       
    365                 370                 375                           
TGC AGA ATA AAA CAA ATC ATA AAC TTG TGG CAG GAA GTA                       
                                1168                                      
Cys Arg Ile Lys Gln Ile Ile Asn Leu Trp Gln Glu Val                       
            380                 385                                       
GGC AAA GCA ATG TAT GCC CCT CCC ATC AGA GGA CAA ATT                       
                                1207                                      
Gly Lys Ala Met Tyr Ala Pro Pro Ile Arg Gly Gln Ile                       
390                 395                 400                               
AGA TGT TCA TCA AAT ATT ACA GGG CTA CTA TTA ACA AGA                       
                                1246                                      
Arg Cys Ser Ser Asn Ile Thr Gly Leu Leu Leu Thr Arg                       
        405                 410                 415                       
GAT GGT GGA AGT AAC ACC GGT GAC AAC AGG ACT GAG ACC                       
                                1285                                      
Asp Gly Gly Ser Asn Thr Gly Asp Asn Arg Thr Glu Thr                       
                420                 425                                   
TTT AGA CCT GGA GGA GGA GAT ATG AGG GAC AAT TGG AGA                       
                                1324                                      
Phe Arg Pro Gly Gly Gly Asp Met Arg Asp Asn Trp Arg                       
    430                 435                 440                           
AGT GAA TTA TAT AAA TAT AAA GTA GTA AGA ATT GAA CCA                       
                                1363                                      
Ser Glu Leu Tyr Lys Tyr Lys Val Val Arg Ile Glu Pro                       
            445                 450                                       
TTA GGA GTA GCA CCC ACC CAG GCA AAG AGA AGA GTG GTG                       
                                1402                                      
Leu Gly Val Ala Pro Thr Gln Ala Lys Arg Arg Val Val                       
455                 460                 465                               
CAA AGA GAA AAA AGA GCA GTG GGG ATA GGA GCT ATG TTC                       
                                1441                                      
Gln Arg Glu Lys Arg Ala Val Gly Ile Gly Ala Met Phe                       
        470                 475                 480                       
CTT GGG TTC TTG GGA GAT AA 1461                                           
Leu Gly Phe Leu Gly Asp                                                   
                 485 486                                                  
CLONE C8.6                                                                
G   GTA CCT GTG TGG AAA GAA GCA ACC ACC ACT CTA TTT                       
                                37                                        
    Val Pro Val Trp Lys Glu Ala Thr Thr Thr Leu Phe                       
      1               5                  10                               
TGT GCA TCA GAT GCT AAA GCA TAT GAT ACA GAG GTA CAT                       
                                76                                        
Cys Ala Ser Asp Ala Lys Ala Tyr Asp Thr Glu Val His                       
         15                  20                  25                       
AAT GTT TGG GCT ACA CAT GCC TGT GTA CCC ACA GAC CCC                       
                                115                                       
Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro                       
                30                  35                                    
AAC CCA CAA GAA GTA GTA TTG GAA AAT GTA ACA GAA AAT                       
                                154                                       
Asn Pro Gln Glu Val Val Leu Glu Asn Val Thr Glu Asn                       
     40                  45                  50                           
TTT AAC ATG TGG AAA AAT AAC ATG GTA GAA CAG ATG CAT                       
                                193                                       
Phe Asn Met Trp Lys Asn Asn Met Val Glu Gln Met His                       
             55                  60                                       
GAG GAT ATA ATC AGT TTA TGG GAT CAA AGT CTA AAG CCA                       
                                232                                       
Glu Asp Ile Ile Ser Leu Trp Asp Gln Ser Leu Lys Pro                       
 65                  70                  75                               
TGT GTA AAA TTA ACC CCA CTC TGT GTT ACT TTA AAT TGC                       
                                271                                       
Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu Asn Cys                       
         80                  85                  90                       
ACT AAT TTG GAG AAT GCT AAT AAT ACC GAG AAT GCT AAT                       
                                310                                       
Thr Asn Leu Glu Asn Ala Asn Asn Thr Glu Asn Ala Asn                       
                 95                 100                                   
AAT ACC AAT AAT TAT ACC TTG GGG ATG GAG AGA GGT GAA                       
                                349                                       
Asn Thr Asn Asn Tyr Thr Leu Gly Met Glu Arg Gly Glu                       
    105                 110                 115                           
AGA AAA AAC TGC TCT TTC AAT ATC ACC ACA AGC TTA AGA                       
                                 388                                      
Arg Lys Asn Cys Ser Phe Asn Ile Thr Thr Ser Leu Arg                       
            120                 125                                       
GAT AAG GGG AAA AAA GAA TAT GCA TTG TTT TAT AAA CTT                       
                                 427                                      
Asp Lys Gly Lys Lys Glu Tyr Ala Leu Phe Tyr Lys Leu                       
130                 135                 140                               
GAT GTA GTA CAA ATA GAT AAT AGT ACC AAC TAT AGG CTG                       
                                 466                                      
Asp Val Val Gln Ile Asp Asn Ser Thr Asn Tyr Arg Leu                       
        145                 150                 155                       
ATA AGT TGT AAT ACC TCA GTC ATT ACA CAG GCC TGT CCA                       
                                 505                                      
Ile Ser Cys Asn Thr Ser Val Ile Thr Gln Ala Cys Pro                       
                160                 165                                   
AAG GTA TCC TTT GAG CCA ATT CCC ATA CAT TAT TGT GCC                       
                                 544                                      
Lys Val Ser Phe Glu Pro Ile Pro Ile His Tyr Cys Ala                       
    170                 175                 180                           
CCG GCT GGT TTT GCG ATT CTA AAG TGT AAA GAT AAG AAG                       
                                 583                                      
Pro Ala Gly Phe Ala Ile Leu Lys Cys Lys Asp Lys Lys                       
            185                 190                                       
TTC AAT GGA ACA GGA CCA TGT AAA AAT GTC AGG ACA GTA                       
                                 622                                      
Phe Asn Gly Thr Gly Pro Cys Lys Asn Val Arg Thr Val                       
195                 200                 205                               
CAA TGT ACA CAT GGA ATT AGA CCA GTA GTA TCA ACT CAA                       
                                 661                                      
Gln Cys Thr His Gly Ile Arg Pro Val Val Ser Thr Gln                       
        210                 215                 220                       
CTA CTG TTA AAT GGC AGT CTA GCA GAA GAA GAG ATA GTA                       
                                 700                                      
Leu Leu Leu Asn Gly Ser Leu Ala Glu Glu Glu Ile Val                       
                225                 230                                   
ATT AGA TCT GAA AAT ATC ACA GAC AAT GCT AAA ACC ATA                       
                                 739                                      
Ile Arg Ser Glu Asn Ile Thr Asp Asn Ala Lys Thr Ile                       
    235                 240                 245                           
ATA GTG CAG CTA AAT GAA TCT ATA GTG ATT AAT TGT ACA                       
                                 778                                      
Ile Val Gln Leu Asn Glu Ser Ile Val Ile Asn Cys Thr                       
            250                 255                                       
AGA CCC AAT AAC AAC ACA AGA AAA AGT ATA AAT ATA GGA                       
                                 817                                      
Arg Pro Asn Asn Asn Thr Arg Lys Ser Ile Asn Ile Gly                       
260                 265                 270                               
CCA GGG AGA GCA TTC TAT ACA ACA GGA GAC ATA ATA GGA                       
                                 856                                      
Pro Gly Arg Ala Phe Tyr Thr Thr Gly Asp Ile Ile Gly                       
        275                 280                 285                       
GAT ATA AGA CAA GCA CAT TGT AAC CTT AGT AAA ACA CAA                       
                                 895                                      
Asp Ile Arg Gln Ala His Cys Asn Leu Ser Lys Thr Gln                       
                290                 295                                   
TGG GAA AAA ACG TTA AGA CAG ATA GCT ATA AAA TTA GAA                       
                                 934                                      
Trp Glu Lys Thr Leu Arg Gln Ile Ala Ile Lys Leu Glu                       
    300                 305                 310                           
GAA AAA TTT AAG AAT AAA ACA ATA GCC TTT AAT AAA TCC                       
                                 973                                      
Glu Lys Phe Lys Asn Lys Thr Ile Ala Phe Asn Lys Ser                       
            315                 320                                       
TCA GGA GGG GAC CCA GAA ATT GTA ATG CAC AGT TTT AAT                       
                                 1012                                     
Ser Gly Gly Asp Pro Glu Ile Val Met His Ser Phe Asn                       
325                 330                 335                               
TGT GGA GGG GGA TTT TTC TAC TGT AGT ACG AGA AAA CTG                       
                                1051                                      
Cys Gly Gly Gly Phe Phe Tyr Cys Ser Thr Arg Lys Leu                       
        340                 345                 350                       
TTT AAT AGT ACC TGG AAT TTA ACA CAA CCG TTT AGT AAT                       
                                 1090                                     
Phe Asn Ser Thr Trp Asn Leu Thr Gln Pro Phe Ser Asn                       
                355                 360                                   
ACC GGG GAT CGT ACT GAA GAG TTA AAT ATT ACA CTC CCA                       
                                 1129                                     
Thr Gly Asp Arg Thr Glu Glu Leu Asn Ile Thr Leu Pro                       
    365                 370                 375                           
TGC AGA ATA AAA CAA ATC ATA AAC TTG TGG CAG GAA GTA                       
                                 1168                                     
Cys Arg Iie Lys Gln Ile Ile Asn Leu Trp Gln Glu Val                       
            380                 385                                       
GGC AAA GCA ATG TAT GCC CCT CCC ATC AGA GGA CAA ATT                       
                                 1207                                     
Gly Lys Ala Met Tyr Ala Pro Pro Ile Arg Gly Gln Ile                       
390                 395                 400                               
AGA TGT TCA TCA AAT ATT ACA GGG CTA CTA TTA AGG AGA                       
                                 1246                                     
Arg Cys Ser Ser Asn Ile Thr Gly Leu Leu Leu Arg Arg                       
        405                 410                 415                       
GAT GGT GGA AGT AAC ACC AGT GAC AAC CAG ACT GAG ACC                       
                                 1285                                     
Asp Gly Gly Ser Asn Thr Ser Asp Asn Gln Thr Glu Thr                       
                420                 425                                   
TTT AGA CCT GGG GGA GGA GAT ATG AGG GAC AAG TGG AGA                       
                                 1324                                     
Phe Arg Pro Gly Gly Gly Asp Met Arg Asp Lys Trp Arg                       
    430                 435                 440                           
AGT GAA TTA TAT AAA TAT AAA GTA GTA AGA ATT GAA CCA                       
                                 1363                                     
Ser Glu Leu Tyr Lys Tyr Lys Val Val Arg Ile Glu Pro                       
            445                 450                                       
TTA GGA GTA GCA CCC ACC CAG GCA AAG AGA AGA GTG GTG                       
                                 1402                                     
Leu Gly Val Ala Pro Thr Gln Ala Lys Arg Arg Val Val                       
455                 460                 465                               
CAA AGA GAA AAA AGA GCA GTG GGG ATA GGA GCT ATG TTC                       
                                 1441                                     
Gln Arg Glu Lys Arg Ala Val Gly Ile Gly Ala Met Phe                       
        470                 475                 480                       
CTT AGG TTC TTA GGA GAT AAA GCT TCT AGA GTC 1474                          
Leu Arg Phe Leu Gly Asp Lys Ala Ser Arg Val                               
                485                 490 491                               
CLONE C15.2                                                               
    CTC GAG GTA CCT GTA TGG AAA GAA GCA ACT ACC ACT                       
                                36                                        
    Leu Glu Val Pro Val Trp Lys Glu Ala Thr Thr Thr                       
      1               5                  10                               
CTA TTT TGT GCA TCA GAT GCT AAA GCA TAT AAT ACA GAG                       
                                 75                                       
Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Asn Thr Glu                       
         15                  20                  25                       
AAA CAT AAT GTT TGG GCC ACA CAC GCC TGT GTA CCC ACA                       
                                 114                                      
Lys His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr                       
GAT CCC AAC CCA CAA GAA GTA GTA TTG GGA AAT GTG ACA                       
                                 153                                      
Asp Pro Asn Pro Gln Glu Val Val Leu Gly Asn Val Thr                       
GAA AAT TTT AAC ATG TGG AAA AAT AAC ATG GTA GAA CAA                       
                                 192                                      
Glu Asn Phe Asn Met Trp Lys Asn Asn Met Val Glu Gln                       
             55                  60                                       
ATG CAT GAA GAT ATA ATC AGT TTA TGG GAT CAA AGT CTA                       
                                 231                                      
Met His Glu Asp Ile Ile Ser Leu Trp Asp Gln Ser Leu                       
 65                  70                  75                               
AAG CCA TGT GTA AAA TTA ACC CCA CTC TGT GTT ACT TTA                       
                                 270                                      
Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu                       
         80                  85                  90                       
AAT TGC ACT GAT GAT TTA GGG AAT GCT ACT AAT ACC AAT                       
                                 309                                      
Asn Cys Thr Asp Asp Leu Gly Asn Ala Thr Asn Thr Asn                       
                 95                 100                                   
AGT AGT GCC ACT ACC AAT AGT AGT AGT TGG GAA GAA ATG                       
                                 348                                      
Ser Ser Ala Thr Thr Asn Ser Ser Ser Trp Glu Glu Met                       
    105                 110                 115                           
AAG GGG GAA ATG AAA AGA TGC TCT TTC AAT ATC ACC ACA                       
                                 387                                      
Lys Gly Glu Met Lys Arg Cys Ser Phe Asn Ile Thr Thr                       
            120                 125                                       
AGC ATA AGA GAT AAG ATT AAG AAA GAA CAT GCA CTT TTC                       
                                 426                                      
Ser Ile Arg Asp Lys Ile Lys Lys Glu His Ala Leu Phe                       
130                 135                 140                               
TAT AGA CTT GAT GTA GTA CCA ATA GAT AAT GAT AAT ACC                       
                                465                                       
Tyr Arg Leu Asp Val Val Pro Ile Asp Asn Asp Asn Thr                       
         145                 150                 155                      
ACA TAT AGG TTG ATA AAT TGT AAT ACC TCA GTC ATT ACA                       
                                504                                       
Thr Tyr Arg Leu Ile Asn Cys Asn Thr Ser Val Ile Thr                       
                160                 165                                   
CAG GCC TGT CCA AAG GTA TCA TTT GAG CCA ATT CCC ATA                       
                                 543                                      
Gln Ala Cys Pro Lys Val Ser Phe Glu Pro Ile Pro Ile                       
    170                 175                 180                           
CAT TTT TGT GCC CCG GCT GGT TTT GCG ATT CTA AAG TGT                       
                                 582                                      
His Phe Cys Ala Pro Ala Gly Phe Ala Ile Leu Lys Cys                       
            185                 190                                       
AAT AAT AAG ACG TTC GAG GGA AAA GGA CCA TGT AAA AAT                       
                                 621                                      
Asn Asn Lys Thr Phe Glu Gly Lys Gly Pro Cys Lys Asn                       
195                 200                 205                               
GTC AGT ACA GTA CAA TGC ACA CAT GGA ATT AGG CCA GTA                       
                                660                                       
Val Ser Thr Val Gln Cys Thr His Gly Ile Arg Pro Val                       
        210                 215                 220                       
GTG TCA ACT CAA CTG CTG TTA AAT GGC AGT CTA GCA GAA                       
                                699                                       
Val Ser Thr Gln Leu Leu Leu Asn Gly Ser Leu Ala Glu                       
                 225                 230                                  
GAA GAG GTA ATA ATT AGA TCT GAC AAT ATC ACA GAC AAT                       
                                738                                       
Glu Glu Val Ile Ile Arg Ser Asp Asn Ile Thr Asp Asn                       
    235                 240                 245                           
ACT AAA ACC ATT ATA GTA CAG CTA AAC GAA TCT GTA GTA                       
                                777                                       
Thr Lys Thr Ile Ile Val Gln Leu Asn Glu Ser Val Val                       
            250                 255                                       
ATT AAT TGT ACA AGA CCC AAC AAC AAT ACA AGA AAA AGT                       
                                816                                       
Ile Asn Cys Thr Arg Pro Asn Asn Asn Thr Arg Lys Ser                       
260                 265                 270                               
ATA CAT ATA GGA CCA GGG AGT GCA TTT TTT GCA ACA GGA                       
                                855                                       
Ile His Ile Gly Pro Gly Ser Ala Phe Phe Ala Thr Gly                       
        275                 280                 285                       
GAA ATA ATA GGA GAT ATA AGA CAA GCA CAC TGT AAC CTT                       
                                894                                       
Glu Ile Ile Gly Asp Ile Arg Gln Ala His Cys Asn Leu                       
                290                 295                                   
AGT AGA ACA CAA TGG AAT AAC ACT TTA GGA AAG ATA GTC                       
                                933                                       
Ser Arg Thr Gln Trp Asn Asn Thr Leu Gly Lys Ile Val                       
    300                 305                 310                           
ATA AAA TTA AGA GAA CAA TTT AGA AAA CAA TTT GGA GAA                       
                                972                                       
Ile Lys Leu Arg Glu Gln Phe Arg Lys Gln Phe Gly Glu                       
            315                 320                                       
AAA ACA ATA GTC TTT AAT CGA TCC TCA GGA GGG GAC CCG                       
                                1011                                      
Lys Thr Ile Val Phe Asn Arg Ser Ser Gly Gly Asp Pro                       
325                 330                 335                               
GAA ATT GCA ATG CAC AGT TTT AAT TGT GGA GGG GAA TTT                       
                                1050                                      
Glu Ile Ala Met His Ser Phe Asn Cys Gly Gly Glu Phe                       
        340                 345                 350                       
TTC TAC TGT AAC ACA ACA GCA CTG TTT AAT AGT ACC TGG                       
                                1089                                      
Phe Tyr Cys Asn Thr Thr Ala Leu Phe Asn Ser Thr Trp                       
                355                 360                                   
AAT GTT ACT AAA GGG TTG AAT AAC ACT GAA GGA AAT AGC                       
                                1128                                      
Asn Val Thr Lys Gly Leu Asn Asn Thr Glu Gly Asn Ser                       
    365                 370                 375                           
ACA GGA GAT GAA AAT ATC ATA CTC CCA TGT AGA ATA AAA                       
                                1167                                      
Thr Gly Asp Glu Asn Ile Ile Leu Pro Cys Arg Ile Lys                       
            380                 385                                       
CAA ATT ATA AAC ATG TGG CAG GAA GTA GGA AAA GCA ATG                       
                                1206                                      
Gln Ile Ile Asn Met Trp Gln Glu Val Gly Lys Ala Met                       
390                 395                 400                               
TAT GCC CCT CCC ATC AGT GGA CAA ATT AGA TGT TCA TCA                       
                                1245                                      
Tyr Ala Pro Pro Ile Ser Gly Gln Ile Arg Cys Ser Ser                       
        405                 410                 415                       
AAC ATT ACA GGG CTG CTA CTA ACA AGA GAT GGT GGT AGT                       
                                1284                                      
Asn Ile Thr Gly Leu Leu Leu Thr Arg Asp Gly Gly Ser                       
                420                 425                                   
AAG AAC GAG AGC ATC ACC ACC GAG GTC TTC AGA CCT GGA                       
                                1323                                      
Lys Asn Glu Ser Ile Thr Thr Glu Val Phe Arg Pro Gly                       
    430                 435                 440                           
GGA GGA GAT ATG AGG GAC AAT TGG AGA AGT GAA TTA TAT                       
                                1362                                      
Gly Gly Asp Met Arg Asp Asn Trp Arg Ser Glu Leu Tyr                       
            445                 450                                       
AAA TAT AAA GTA GTA AAA ATT GAA CCA TTA GGA GTA GCG                       
                                1401                                      
Lys Tyr Lys Val Val Lys Ile Glu Pro Leu Gly Val Ala                       
455                 460                 465                               
CCC ACC AAG GCA AAG AGA AGA GTG GTG CAG AGA GAA AAA                       
                                1440                                      
Pro Thr Lys Ala Lys Arg Arg Val Val Gln Arg Glu Lys                       
        470                 475                 480                       
AGA GCA GTG GGA ACA ATA GGA GCT ATG TTC CTT GGG TTC                       
                                1479                                      
Arg Ala Val Gly Thr Ile Gly Ala Met Phe Leu Gly Phe                       
                485                 490                                   
TTG GGA GCA TAA AGC TTC TAG AGT CGA CCT GCA 1512                          
Leu Gly Ala Xaa Ser Phe Xaa Ser Arg Pro Ala                               
    495                 500             504                               
CLONE C15.3                                                               
    CTC GAG GTA CCT GTG TGG AAA GAA GCA ACT ACC ACT                       
                                36                                        
    Leu Glu Val Pro Val Trp Lys Glu Ala Thr Thr Thr                       
      1               5                  10                               
CTA TTT TGT GCA TCA GAT GCT AAA GCA TAT AAT ACA GAG                       
                                75                                        
Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Asn Thr Glu                       
         15                  20                  25                       
AAA CAT AAT GTT TGG GCC ACA CAC GCC TGT GTA CCC ACA                       
                                114                                       
Lys His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr                       
                 30                  35                                   
GAT CCC AAC CCA CAA GAA GTA GTA TTG GGA AAT GTG ACA                       
                                153                                       
Asp Pro Asn Pro Gln Glu Val Val Leu Gly Asn Val Thr                       
     40                  45                 50                            
GAA AAT TTT AAC ATG TGG AAA AAT AAC ATG GTA GAA CAA                       
                                192                                       
Glu Asn Phe Asn Met Trp Lys Asn Asn Met Val Glu Gln                       
             55                  60                                       
ATG CAT GAA GAT ATA ATC AGT TTA TGG GAT CAA AGT CTA                       
                                231                                       
 Met His Glu Asp Ile Ile Ser Leu Trp Asp Gln Ser Leu                      
  65                  70                  75                              
AAG CCA TGT GTA AAA TTA ACC CCA CTC TGT GTT ACT TTA                       
                                270                                       
Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu                       
         80                  85                  90                       
AAT TGC ACT GAT GAT TTA GGG AAT GCT ACT AAT ACC AAT                       
                                309                                       
Asn Cys Thr Asp Asp Leu Gly Asn Ala Thr Asn Thr Asn                       
                 95                 100                                   
AGC AGT GCC ACT ACC AAT AGT AGT AGT TGG GAA GAA ATG                       
                                348                                       
Ser Ser Ala Thr Thr Asn Ser Ser Ser Trp Glu Glu Met                       
    105                 110                 115                           
AAG GGG GAA ATG AAA AGG TGC TCT TTC AAT ATC ACC ACA                       
                                387                                       
Lys Gly Glu Met Lys Arg Cys Ser Phe Asn Ile Thr Thr                       
            120                 125                                       
AGC ATA AGA GAT AAG ATT AAG AAA GAA CAT GCA CTT TTC                       
                                426                                       
Ser Ile Arg Asp Lys Ile Lys Lys Glu His Ala Leu Phe                       
130                 135                 140                               
TAT AGA CTT GAT GTA GTA CCA ATA GAT AAT GAT AAT ACC                       
                                465                                       
Tyr Arg Leu Asp Val Val Pro Ile Asp Asn Asp Asn Thr                       
        145                 150                 155                       
ACA TAT AGG TTG ATA AAT TGT AAT ACC TCA GTC ATT ACA                       
                                504                                       
Thr Tyr Arg Leu Ile Asn Cys Asn Thr Ser Val Ile Thr                       
                160                 165                                   
CAG GCC TGT CCA AAG GTA TCA TTT GAG CCA ATT CCC ATA                       
                                543                                       
Gln Ala Cys Pro Lys Val Ser Phe Glu Pro Ile Pro Ile                       
    170                 175                 180                           
CAT TTT TGT GCC CCG GCT GGT TTT GCG ATT CTA AAG TGT                       
                                582                                       
His Phe Cys Ala Pro Ala Gly Phe Ala Ile Leu Lys Cys                       
            185                 190                                       
AAT AAT AAG ACG TTC GAG GGA AAA GGA CCA TGT AAA AAT                       
                                621                                       
Asn Asn Lys Thr Phe Glu Gly Lys Gly Pro Cys Lys Asn                       
195                 200                 205                               
GTC AGT ACA GTA CAA TGC ACA CAT GGA ATT AGG CCA GTA                       
                                660                                       
Val Ser Thr Val Gln Cys Thr His Gly Ile Arg Pro Val                       
        210                 215                 220                       
GTG TCA ACT CAA CTG CTG TTA AAT GGC AGT CTA GCA GAA                       
                                699                                       
Val Ser Thr Gln Leu Leu Leu Asn Gly Ser Leu Ala Glu                       
                225                 230                                   
GAA GAG GTA ATA ATT AGA TCT GGC AAT ATC ACA GAC AAT                       
                                738                                       
Glu Glu Val Ile Ile Arg Ser Gly Asn Ile Thr Asp Asn                       
    235                 240                 245                           
ACT AAA ACC ATT ATA GTA CAG CTA AAC GAA TCT GTA GTA                       
                                777                                       
Thr Lys Thr Ile Ile Val Gln Leu Asn Glu Ser Val Val                       
            250                 255                                       
ATT AAT TGT ACA AGA TCC AAC AAC AAT ACA AGA AAA AGT                       
                                816                                       
Ile Asn Cys Thr Arg Ser Asn Asn Asn Thr Arg Lys Ser                       
260                 265                 270                               
ATA CAT ATA GGA CCA GGG AGT GCA TTT TTT GCA ACA GGA                       
                                855                                       
Ile His Ile Gly Pro Gly Ser Ala Phe Phe Ala Thr Gly                       
        275                 280                 285                       
GAA ATA ATA GGA GAT ATA AGA CAA GCA CAC TGT AAC CTT                       
                                894                                       
Glu Ile Ile Gly Asp Ile Arg Gln Ala His Cys Asn Leu                       
                290                 295                                   
AGT AGA ACA CAA TGG AAT AAC ACT TTA GGA AAG ATA GTC                       
                                933                                       
Ser Arg Thr Gln Trp Asn Asn Thr Leu Gly Lys Ile Val                       
    300                 305                 310                           
ATA AAA TTA AGA GAA CAA TTT AGA AAA CAA TTT GGA GAA                       
                                972                                       
Ile Lys Leu Arg Glu Gln Phe Arg Lys Gln Phe Gly Glu                       
            315                 320                                       
AAA ACA ATA GTC TTT AAT CGA TCC TCA GGA GGG GAC CCG                       
                                1011                                      
Lys Thr Ile Val Phe Asn Arg Ser Ser Gly Gly Asp Pro                       
325                 330                 335                               
GAA ATT GCA ATG CAC AGT TTT AAT TGT GGA GGG GAA TTT                       
                                1050                                      
Glu Ile Ala Met His Ser Phe Asn Cys Gly Gly Glu Phe                       
        340                 345                 350                       
TTC TAC TGT AAC ACA ACA GCA CTG TTT AAT AGT ACC TGG                       
                                1089                                      
Phe Tyr Cys Asn Thr Thr Ala Leu Phe Asn Ser Thr Trp                       
                355                 360                                   
AAT GTT ACT AAA GGG TTG AAT AAC ACT GAA GGA AAT AGC                       
                                1128                                      
Asn Val Thr Lys Gly Leu Asn Asn Thr Glu Gly Asn Ser                       
    365                 370                 375                           
ACA GGG GAT GAA AAT ATC ATA CTC CCA TGT AGA ATA AAA                       
                                1167                                      
Thr Gly Asp Glu Asn Ile Ile Leu Pro Cys Arg Ile Lys                       
            380                 385                                       
CAA ATT ATA AAC ATG TGG CAG GAA GTA GGA AAA GCA ATG                       
                                1206                                      
Gln Ile Ile Asn Met Trp Gln Glu Val Gly Lys Ala Met                       
390                 395                 400                               
TAT GCC CCT CCC ATC AGT GGA CAA ATT AGA TGT TCA TCA                       
                                1245                                      
Tyr Ala Pro Pro Ile Ser Gly Gln Ile Arg Cys Ser Ser                       
        405                 410                 415                       
AAT ATT ACA GGG CTG CTA CTA ACA AGA GAT GGT GGT AGT                       
                                1284                                      
Asn Ile Thr Gly Leu Leu Leu Thr Arg Asp Gly Gly Ser                       
                420                 425                                   
AAG AAC GAG AGC ATC ACC ACC GAG GTC TTC AGA CCT GGA                       
                                1323                                      
Lys Asn Glu Ser Ile Thr Thr Glu Val Phe Arg Pro Gly                       
    430                 435                 440                           
GGA GGA GAT ATG AGG GAC AAT TGG AGA AGT GAA TTA TAT                       
                                1362                                      
Gly Gly Asp Met Arg Asp Asn Trp Arg Ser Glu Leu Tyr                       
            445                 450                                       
AAA TAT AAA GTA GTA AAA ATT GAA CCA TTA GGA GTA GCG                       
                                1401                                      
Lys Tyr Lys Val Val Lys Ile Glu Pro Leu Gly Val Ala                       
455                 460                 465                               
CCC ACC AAG GCA AAG AGA AGA GTG GTG CAG AGA GAA AAA                       
                                1440                                      
Pro Thr Lys Ala Lys Arg Arg Val Val Gln Arg Glu Lys                       
        470                 475                 480                       
AGA GCA GTG GGA ACA ATA GGA GCT ATG TTC CTT GGG TTC                       
                                1479                                      
Arg Ala Val Gly Thr Ile Gly Ala Met Phe Leu Gly Phe                       
                485                 490                                   
TTA GGA GCA TAA AGC TTC TAG A 1501                                        
Leu Gly Ala Xaa Ser Phe Xaa                                               
    495                 500                                               
CLONE C7.2                                                                
GG GAA TTC GGA TCC GGG GTA CCT GTG TGG AAG GAA GCA                        
                                38                                        
   Glu Phe Gly Ser Gly Val Pro Val Trp Lys Glu Ala                        
     1               5                  10                                
ACC ACC ACT CTA TTC TGT GCA TCA GAT GCT AGA GCA TAT                       
                                77                                        
Thr Thr Thr Leu Phe Cys Ala Ser Asp Ala Arg Ala Tyr                       
         15                  20                  25                       
GAC ACA GAG GTA CAT AAT GTT TGG GCC ACA CAT GCC TGT                       
                                116                                       
Asp Thr Glu Val His Asn Val Trp Ala Thr His Ala Cys                       
                 30                  35                                   
GTA CCC ACA GAC CCT AGT CCA CAA GAA GTA GTT TTG GAA                       
                                155                                       
Val Pro Thr Asp Pro Ser Pro Gln Glu Val Val Leu Glu                       
     40                  45                  50                           
AAT GTG ACA GAA AAT TTT AAC ATG TGG AAA AAT AAC ATG                       
                                194                                       
Asn Val Thr Glu Asn Phe Asn Met Trp Lys Asn Asn Met                       
             55                  60                                       
GTA GAA CAA ATG CAT GAG GAT ATA ATT AGT TTA TGG GAT                       
                                233                                       
Val Glu Gln Met His Glu Asp Ile Ile Ser Leu Trp Asp                       
 65                  70                  75                               
CAA AGC TTA AAG CCA TGT GTA AAA TTA ACC CCA CTC TGT                       
                                272                                       
Gln Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys                       
         80                  85                  90                       
GTT ACT TTA AAT TGC AGT GAT TAT AGG AAT GCT ACT GAT                       
                                311                                       
Val Thr Leu Asn Cys Ser Asp Tyr Arg Asn Ala Thr Asp                       
                 95                 100                                   
TAT AAG AAT GCT ACT GAT ACC ACT AGT AGT AAC GAG GGA                       
                                350                                       
Tyr Lys Asn Ala Thr Asp Thr Thr Ser Ser Asn Glu Gly                       
    105                 110                 115                           
AAG ATG GAG AGA GGA GAA ATA AAA AAC TGC TCT TTC AAT                       
                                389                                       
Lys Met Glu Arg Gly Glu Ile Lys Asn Cys Ser Phe Asn                       
            120                 125                                       
ATT ACC ACA AGC ATA AAA AAT AAG ATG CAG AAA GAA TAT                       
                                428                                       
Ile Thr Thr Ser Ile Lys Asn Lys Met Gln Lys Glu Tyr                       
130                 135                 140                               
GCA CTT TTC TAT AAA CTT GAT ATA GTA CCA ATA GAT AAT                       
                                467                                       
Ala Leu Phe Tyr Lys Leu Asp Ile Val Pro Ile Asp Asn                       
        145                 150                 155                       
ACA AGC TAT ACA TTG ATA AGT TGT AAC ACC TCA GTC ATT                       
                                506                                       
Thr Ser Tyr Thr Leu Ile Ser Cys Asn Thr Ser Val Ile                       
                160                 165                                   
ACA CAG GCC TGT CCA AAG GTA TCC TTT GAA CCA ACT CCC                       
                                545                                       
Thr Gln Ala Cys Pro Lys Val Ser Phe Glu Pro Thr Pro                       
    170                 175                 180                           
ATA CAT TAT TGT GCT CCG GCT GGT TTT GCG ATT CTA AAG                       
                                584                                       
Ile His Tyr Cys Ala Pro Ala Gly Phe Ala Ile Leu Lys                       
            185                 190                                       
TGT AAT GAT AAG AAG TTC AGT GGA AAA GGA GAA TGT AAA                       
                                623                                       
Cys Asn Asp Lys Lys Phe Ser Gly Lys Gly Glu Cys Lys                       
195                 200                 205                               
AAT GTC AGC ACA GTA CAA TGT ACA CAT GGA ATT AGG CCA                       
                                662                                       
Asn Val Ser Thr Val Gln Cys Thr His Gly Ile Arg Pro                       
        210                 215                 220                       
GTA GTA TCA ACT CAA CTG CTG TTA AAT GGC AGT CTA GCA                       
                                701                                       
Val Val Ser Thr Gln Leu Leu Leu Asn Gly Ser Leu Ala                       
                225                 230                                   
GAA GAA GAG GTG GTA ATT AGA TCT GAC AAT TTC ATA GAC                       
                                740                                       
Glu Glu Glu Val Val Ile Arg Ser Asp Asn Phe Ile Asp                       
    235                240                  245                           
AAT ACT AAA ACC ATA ATA GTA CAG CTG AAA GAA TCT GTA                       
                                779                                       
Asn Thr Lys Thr Ile Ile Val Gln Leu Lys Glu Ser Val                       
            250                 255                                       
GAA ATT AAT TGT ATA AGA CCC AAC AAT AAT ACA AGA AAA                       
                                818                                       
Glu Ile Asn Cys Ile Arg Pro Asn Asn Asn Thr Arg Lys                       
260                 265                 270                               
GGT ATA CAT ATA GGA CCA GGG AGA GCA TGG TAT GCA ACA                       
                                857                                       
Gly Ile His Ile Gly Pro Gly Arg Ala Trp Tyr Ala Thr                       
        275                 280                 285                       
GGA GAA ATA GTA GGA GAT ATA AGA AAG GCA TAT TGT AAC                       
                                896                                       
Gly Glu Ile Val Gly Asp Ile Arg Lys Ala Tyr Cys Asn                       
                290                 295                                   
ATT AGT AGA ACA AAA TGG AAT AAC ACT TTA ATA CAG ATA                       
                                935                                       
Ile Ser Arg Thr Lys Trp Asn Asn Thr Leu Ile Gln Ile                       
    300                 305                 310                           
GCT AAC AAA TTA AAA GAA AAA TAT AAT ACA ACA ATA AGC                       
                                974                                       
Ala Asn Lys Leu Lys Glu Lys Tyr Asn Thr Thr Ile Ser                       
            315                 320                                       
TTT AAT CGA TCC TCA GGA GGG GAC CCA GAA ATT GTA ACG                       
                                1013                                      
Phe Asn Arg Ser Ser Gly Gly Asp Pro Glu Ile Val Thr                       
325                 330                 335                               
CAT AGT TTT AAT TGT GGA GGG GAG TTT TTC TAC TGT GAT                       
                                1052                                      
His Ser Phe Asn Cys Gly Gly Glu Phe Phe Tyr Cys Asp                       
        340                 345                 350                       
TCA ACA CAA CTG TTT AAT AGT ACT TGG AAT TTA AAT GGT                       
                                1091                                      
Ser Thr Gln Leu Phe Asn Ser Thr Trp Asn Leu Asn Gly                       
                355                 360                                   
ACT TGG AAT TTT ACT GCA GGG TCA AAT GAA ACT GAA GGC                       
                                1130                                      
Thr Trp Asn Phe Thr Ala Gly Ser Asn Glu Thr Glu Gly                       
    365                 370                 375                           
AAT ATC ACA CTC CCA TGC AGA ATA AAA CAA ATT ATA AAC                       
                                1169                                      
Asn Ile Thr Leu Pro Cys Arg Ile Lys Gln Ile Ile Asn                       
            380                 385                                       
AGG TGG CAG GAA GTA GGG AAA GCA ATG TAT GCC CCT CCC                       
                                1208                                      
Arg Trp Gln Glu Val Gly Lys Ala Met Tyr Ala Pro Pro                       
390                 395                 400                               
ATC AGT GGA CAA ATA AAA TGC TCA TCA AAC ATT ACA GGG                       
                                1247                                      
Ile Ser Gly Gln Ile Lys Cys Ser Ser Asn Ile Thr Gly                       
        405                 410                 415                       
ATG ATA TTA ACA AGG GAT GGT GGT AAC GAG AAC AAT AAT                       
                                1286                                      
Met Ile Leu Thr Arg Asp Gly Gly Asn Glu Asn Asn Asn                       
                420                 425                                   
GAG AGC AGT ACT ACT GAG ACC TTC AGA CCG GGA GGA GGA                       
                                1325                                      
Glu Ser Ser Thr Thr Glu Thr Phe Arg Pro Gly Gly Gly                       
    430                 435                 440                           
GAT ATG AGG AAC AAT TGG AGA AGT GAA TTA TAT AAA TAT                       
                                1364                                      
Asp Met Arg Asn Asn Trp Arg Ser Glu Leu Tyr Lys Tyr                       
            445                 450                                       
AAA GTA GTA AAA ATT GAA CCA TTA GGA GTA GCA CCC ACC                       
                                1403                                      
Lys Val Val Lys Ile Glu Pro Leu Gly Val Ala Pro Thr                       
455                 460                 465                               
AAG GCA AAG AGA AGA GTG GTG CAG AGA GAA AAA AGA GCA                       
                                1442                                      
Lys Ala Lys Arg Arg Val Val Gln Arg Glu Lys Arg Ala                       
        470                 475                 480                       
GTG GGA GCG CTA GGA GCT ATG TTC CTT GGG TTC TTA GGA                       
                                1481                                      
Val Gly Ala Leu Gly Ala Met Phe Leu Gly Phe Leu Gly                       
                485                 490                                   
GCA TAA AGC TTC TAG ACC GAC TCT AGA GGA TCC 1514                          
Ala Xaa Ser Phe Xaa Thr Asp Ser Arg Gly Ser                               
    495                 500             504                               
CLONE C7.10                                                               
G   GTA CCT GTG TGG AAG GAA GCA ACC ACC ACT CTA TTC                       
                                37                                        
    Val Pro Val Trp Lys Glu Ala Thr Thr Thr Leu Phe                       
      1               5                  10                               
TGT GCA TCA GAT GCT AGA GCA TAT GAC ACA GAG GTA CAT                       
                                76                                        
Cys Ala Ser Asp Ala Arg Ala Tyr Asp Thr Glu Val His                       
         15                  20                  25                       
AAT GTT TGG GCC ACA CAT GCC TGT GTA CCC ACA GAC CCT                       
                                115                                       
Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro                       
                 30                  35                                   
AGT CCA CAA GAA GTA TTT TTG GGA AAT GTG ACA GAA AAT                       
                                154                                       
Ser Pro Gln Glu Val Phe Leu Gly Asn Val Thr Glu Asn                       
     40                  45                  50                           
TTT AAT ATG TGG AAA AAT AAC ATG GTA GAA CAA ATG TAT                       
                                193                                       
Phe Asn Met Trp Lys Asn Asn Met Val Glu Gln Met Tyr                       
             55                  60                                       
GAG GAT ATA ATT AGT TTA TGG GAT CAA AGC TTA AAG CCA                       
                                232                                       
Glu Asp Ile Ile Ser Leu Trp Asp Gln Ser Leu Lys Pro                       
 65                  70                  75                               
TGT GTA AAA TTA ACC CCA CTC TGT GTT ACT TTA AAT TGC                       
                                271                                       
Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu Asn Cys                       
         80                  85                  90                       
AGT GAT TAT AGG AAT GCT ACT GAT TAT AAG AAT GCT ACT                       
                                310                                       
Ser Asp Tyr Arg Asn Ala Thr Asp Tyr Lys Asn Ala Thr                       
                 95                 100                                   
GAT ACC ACT AGT AGT AAC GAG GGA AAG ATG GAG AGA GGA                       
                                349                                       
Asp Thr Thr Ser Ser Asn Glu Gly Lys Met Glu Arg Gly                       
    105                 110                 115                           
GAA ATA AAA AAC TGC TCT TTC AAT ATC ACC ACA AGC ATA                       
                                388                                       
Glu Ile Lys Asn Cys Ser Phe Asn Ile Thr Thr Ser Ile                       
            120                 125                                       
AAA AAT AAG ATG CAG AAA GAA TAT GCA CTT TTC TAT AAA                       
                                427                                       
Lys Asn Lys Met Gln Lys Glu Tyr Ala Leu Phe Tyr Lys                       
130                 135                 140                               
CTT AAT ATA GTA CCA ATA GAT AAT ACA AGC TAT ACA TTG                       
                                466                                       
Leu Asn Ile Val Pro Ile Asp Asn Thr Ser Tyr Thr Leu                       
        145                 150                 155                       
ATA AGT TGT AAC ACC TCA GTC ATT ACA CAG GCC TGT CCA                       
                                505                                       
Ile Ser Cys Asn Thr Ser Val Ile Thr Gln Ala Cys Pro                       
                160                 165                                   
AAG GTA TCC TTT GAA CCA ATT CCC ATA CAT TAT TGT GCT                       
                                544                                       
Lys Val Ser Phe Glu Pro Ile Pro Ile His Tyr Cys Ala                       
    170                 175                 180                           
CCG GCT GGT TTT GCG ATT CTA AAG TGT AAT GAT AAG AAG                       
                                583                                       
Pro Ala Gly Phe Ala Ile Leu Lys Cys Asn Asp Lys Lys                       
            185                 190                                       
TTC AGT GGA AAA GGA GAA TGT AAA AAT GTC AGC ACA GTA                       
                                622                                       
Phe Ser Gly Lys Gly Glu Cys Lys Asn Val Ser Thr Val                       
195                 200                 205                               
CAA TGT ACA CAT GGA ATT AGG CCA GTA GTA TCA ACT CAA                       
                                661                                       
Gln Cys Thr His Gly Ile Arg Pro Val Val Ser Thr Gln                       
        210                 215                 220                       
CTG CTG TTA AAT GGC AGT CTA GCA GAA GAA GAG GTG GTA                       
                                700                                       
Leu Leu Leu Asn Gly Ser Leu Ala Glu Glu Glu Val Val                       
                225                 230                                   
ATT AGA TCT GAC AAT TTC ACA GAC AAT ACT AAA ACC ATA                       
                                739                                       
Ile Arg Ser Asp Asn Phe Thr Asp Asn Thr Lys Thr Ile                       
    235                 240                 245                           
ATA GTA CAG CTG AAA GAA TCT GTA GAA ATT AAT TGT ATA                       
                                778                                       
Ile Val Gln Leu Lys Glu Ser Val Glu Ile Asn Cys Ile                       
            250                 255                                       
AGA CCC AAC AAT AAT ACA AGA AAA GGT ATA CAT ATA GGA                       
                                817                                       
Arg Pro Asn Asn Asn Thr Arg Lys Gly Ile His Ile Gly                       
260                 265                 270                               
CCA GGG AGA GCA TGG TAT GCA ACA GGA GAA ATA GTA GGA                       
                                856                                       
Pro Gly Arg Ala Trp Tyr Ala Thr Gly Glu Ile Val Gly                       
        275                 280                 285                       
GAT ATA AGA CAG GCA TAT TGT AAC ATT AGT AGA ACA AAA                       
                                895                                       
Asp Ile Arg Gln Ala Tyr Cys Asn Ile Ser Arg Thr Lys                       
                290                 295                                   
TGG AAT AAC ACT TTA ATA CAG ATA GCT AAC AAA TTA AAA                       
                                934                                       
Trp Asn Asn Thr Leu Ile Gln Ile Ala Asn Lys Leu Lys                       
    300                 305                 310                           
GAA AAA TAT AAT ACA ACA ATA AGC TTT AAT CGA TCC TCA                       
                                973                                       
Glu Lys Tyr Asn Thr Thr Ile Ser Phe Asn Arg Ser Ser                       
            315                 320                                       
GGA GGG GAC CCA GAA ATT GTA ACC CAT AGT TTT AAT TGT                       
                                1012                                      
Gly Gly Asp Pro Glu Ile Val Thr His Ser Phe Asn Cys                       
325                 330                 335                               
GGA GGG GAA TTT TTC TAC TGT AAT TCA ACA CAA CTG TTT                       
                                1051                                      
Gly Gly Glu Phe Phe Tyr Cys Asn Ser Thr Gln Leu Phe                       
        340                 345                 350                       
AAT AGT ACT TGG AAT TTA AAT GGT ACT TGG AAT TTT ACT                       
                                1090                                      
Asn Ser Thr Trp Asn Leu Asn Gly Thr Trp Asn Phe Thr                       
                355                 360                                   
GCA GGG TCA AAT GAA ACT GAA GGC AAT ATC ACA CTC CCA                       
                                1129                                      
Ala Gly Ser Asn Glu Thr Glu Gly Asn Ile Thr Leu Pro                       
    365                 370                 375                           
TGC AGA ATA AAA CAA ATT ATA AAC AGG TGG CAG GAA GTA                       
                                1168                                      
Cys Arg Ile Lys Gln Ile Ile Asn Arg Trp Gln Glu Val                       
            380                 385                                       
GGA AAA GCA ATG TAT GCC CCT CCC ATC AGT GGA CAA ATA                       
                                1207                                      
Gly Lys Ala Met Tyr Ala Pro Pro Ile Ser Gly Gln Ile                       
390                 395                 400                               
AGA TGC TCA TCA AAC ATT ACA GGG ATG ATA TTA ACA AGG                       
                                1246                                      
Arg Cys Ser Ser Asn Ile Thr Gly Met Ile Leu Thr Arg                       
        405                 410                 415                       
GAT GGT GGT AAC GAG AAC AAT AAT GAG AGC AGT ACT ACT                       
                                1285                                      
Asp Gly Gly Asn Glu Asn Asn Asn Glu Ser Ser Thr Thr                       
                420                 425                                   
GAG ACC TTC AGA CCG GGA GGA GGA GAT ATG AGG AAC AAT                       
                                1324                                      
Glu Thr Phe Arg Pro Gly Gly Gly Asp Met Arg Asn Asn                       
    430                 435                 440                           
TGG AGA AGT GAA TTA TAT AAA TAT AAA GTA GTA AAA ATT                       
                                1363                                      
Trp Arg Ser Glu Leu Tyr Lys Tyr Lys Val Val Lys Ile                       
            445                 450                                       
GAG CCA TTA GGA GTA GCA CCC ACC GAC TCT AGA GGA TCC                       
                                1402                                      
Glu Pro Leu Gly Val Ala Pro Thr Asp Ser Arg Gly Ser                       
455                 460                 465                               
TCT AGA 1408                                                              
Ser Arg                                                                   
    469                                                                   
CLONE C11.5                                                               
    GAG GTA CCT GTG TGG AAA GAA GCA ACC ACT ACT CTA                       
                                36                                        
    Glu Val Pro Val Trp Lys Glu Ala Thr Thr Thr Leu                       
      1               5                 10                                
TTT TGT GCA TCA GAT GCT AAA GCA TAT GAC ACA GGG GTG                       
                                75                                        
Phe Cys Ala Ser Asp Ala Lys Ala Tyr Asp Thr Gly Val                       
         15                  20                  25                       
CAT AAT GTT TGG GCC ACA CAT GCC TGT GTA CCC ACA GAC                       
                                114                                       
His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp                       
                 30                  35                                   
CCC AAC CCA CAA GAA ATA GAA TTG GTA AAT GTG ACA GAA                       
                                153                                       
Pro Asn Pro Gln Glu Ile Glu Leu Val Asn Val Thr Glu                       
     40                  45                  50                           
GAT TTT AAC ATG TGG AAA AAT AAA ATG GTA GAC CAG ATG                       
                                192                                       
Asp Phe Asn Met Trp Lys Asn Lys Met Val Asp Gln Met                       
             55                  60                                       
CAT GAG GAT ATA ATC AGT TTA TGG GAT GAA AGC CTA AAG                       
                                231                                       
His Glu Asp Ile Ile Ser Leu Trp Asp Glu Ser Leu Lys                       
 65                  70                  75                               
CCA TGT GTA AAG TTA ACC CCA CTT TGT GTT ACT CTA AAC                       
                                270                                       
Pro Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu Asn                       
         80                  85                  90                       
TGC AGT GAT GTG AAC AAT TCC ACA AAT CCT AAT GAT ACT                       
                                309                                       
Cys Ser Asp Val Asn Asn Ser Thr Asn Pro Asn Asp Thr                       
                 95                 100                                   
AAT ACT AAT TCC ACT AAT ACT ACT TCC TCT ACT CCT ACG                       
                                348                                       
Asn Thr Asn Ser Thr Asn Thr Thr Ser Ser Thr Pro Thr                       
    105                 110                 115                           
GCC ACT ACT AGT AGC GAG GAA AAG ATG GAG AAG GGA GAA                       
                                387                                       
Ala Thr Thr Ser Ser Glu Glu Lys Met Glu Lys Gly Glu                       
            120                 125                                       
ATA AAA AAC TGC TCT TTC AAT ATC ACC ACA CAC ATG AAA                       
                                426                                       
Ile Lys Asn Cys Ser Phe Asn Ile Thr Thr His Met Lys                       
130                 135                 140                               
GAT AAG GCA CAG AAA GAA TAT GCA CTT TTT TAT AAA CTT                       
                                465                                       
Asp Lys Ala Gln Lys Glu Tyr Ala Leu Phe Tyr Lys Leu                       
        145                 150                 155                       
GAT ATA GTA CCA ATA GAT GAT AAT AAT GCC AGC TAT AGG                       
                                504                                       
Asp Ile Val Pro Ile Asp Asp Asn Asn Ala Ser Tyr Arg                       
                160                 165                                   
TTG ATA AGT TGT AAT ACC TCA GAC ATT ACA CAG GCC TGT                       
                                543                                       
Leu Ile Ser Cys Asn Thr Ser Asp Ile Thr Gln Ala Cys                       
    170                 175                 180                           
CCA AAG GTG ACC TTT GAG CCA ATT CCC ATA CAT TAT TGT                       
                                582                                       
Pro Lys Val Thr Phe Glu Pro Ile Pro Ile His Tyr Cys                       
            185                 190                                       
GCC CCG GCT GGT TTT GCG ATT CTA AAG TGT AAA GAT AAG                       
                                621                                       
Ala Pro Ala Gly Phe Ala Ile Leu Lys Cys Lys Asp Lys                       
195                 200                 205                               
AAG TTC AAT GGA ACA GGA CCA TGT TCA AAG GTC AGC ACA                       
                                660                                       
Lys Phe Asn Gly Thr Gly Pro Cys Ser Lys Val Ser Thr                       
        210                 215                 220                       
GTA CAA TGT ACA CAT GGA ATT AGG CCA GTA GTA TCA ACT                       
                                699                                       
Val Gln Cys Thr His Gly Ile Arg Pro Val Val Ser Thr                       
                225                 230                                   
CAA CTG TTG TTA AAT GGC AGT CTT GCA GAA GAA GAA GTA                       
                                738                                       
Gln Leu Leu Leu Asn Gly Ser Leu Ala Glu Glu Glu Val                       
    235                 240                 245                           
GTA ATT AGA TCT GTC AAT TTC ACA GAC AAT GCT AAA ATC                       
                                777                                       
Val Ile Arg Ser Val Asn Phe Thr Asp Asn Ala Lys Ile                       
            250                 255                                       
ATA ATA GTA CAG CTG AAA GAA CCT GTA GCA ATT AAT TGT                       
                                816                                       
Ile Ile Val Gln Leu Lys Glu Pro Val Ala Ile Asn Cys                       
260                 265                 270                               
ACA AGA CCC AAC AAC AAT ACA AGA AAA GGT ATA CAT CTA                       
                                855                                       
Thr Arg Pro Asn Asn Asn Thr Arg Lys Gly Ile His Leu                       
        275                 280                 285                       
GGA CCA GGG AGC ACA TTT TAT ACA ACA GGA GAA ATA ATA                       
                                894                                       
Gly Pro Gly Ser Thr Phe Tyr Thr Thr Gly Glu Ile Ile                       
                290                 295                                   
GGA GAC ATA AGA AAA GCA TAT TGC AAG ATT AGT AAA GAA                       
                                933                                       
Gly Asp Ile Arg Lys Ala Tyr Cys Lys Ile Ser Lys Glu                       
    300                 305                 310                           
AAA TGG AAT AAC ACT TTA AGA CAG GTA GTT AAA AAA TTA                       
                                972                                       
Lys Trp Asn Asn Thr Leu Arg Gln Val Val Lys Lys Leu                       
            315                 320                                       
AGA GAA CAA TTT GGG AAT AAA ACA ATA ATT TTT AAT CGA                       
                                1011                                      
Arg Glu Gln Phe Gly Asn Lys Thr Ile Ile Phe Asn Arg                       
325                 330                 335                               
TCC TCA GGA GGG GAC CCA GAA ATT GTA ATG CAC AGT TTT                       
                                1050                                      
Ser Ser Gly Gly Asp Pro Glu Ile Val Met His Ser Phe                       
        340                 345                 350                       
AAC TGT GGA GGG GAG TTT TTC TAC TGT AAT ACA ACA CAA                       
                                1089                                      
Asn Cys Gly Gly Glu Phe Phe Tyr Cys Asn Thr Thr Gln                       
                355                 360                                   
CTG TTT AAT AGT ACT TGG AAT AAT ACT GAA GGG ACA AAT                       
                                1128                                      
Leu Phe Asn Ser Thr Trp Asn Asn Thr Glu Gly Thr Asn                       
    365                 370                 375                           
AGC ACT GAA GGA AAT AGC ACA ATC ACA CTC CCA TGC AGA                       
                                1167                                      
Ser Thr Glu Gly Asn Ser Thr Ile Thr Leu Pro Cys Arg                       
            380                 385                                       
ATA AAA CAA ATT ATA AAT ATG TGG CAG GAA GTA GGA AAA                       
                                1206                                      
Ile Lys Gln Ile Ile Asn Met Trp Gln Glu Val Gly Lys                       
390                 395                 400                               
GCA ACG TAT GCC CCT CCC ATC AGA GGA CGA ATT AGA TGC                       
                                1245                                      
Ala Thr Tyr Ala Pro Pro Ile Arg Gly Arg Ile Arg Cys                       
        405                 410                 415                       
ATA TCA AAT ATT ACA GGA CTG CTA TTA ACA AGA GAT GGT                       
                                1284                                      
Ile Ser Asn Ile Thr Gly Leu Leu Leu Thr Arg Asp Gly                       
                420                 425                                   
GGT AGG AAT GTC ACA AAC AAT ACC GAA ACC TTC AGA CCT                       
                                1323                                      
Gly Arg Asn Val Thr Asn Asn Thr Glu Thr Phe Arg Pro                       
    430                 435                 440                           
GGA GGA GGA GAC ATG AGG GAC AAT TGG AGA AGT GAA TTA                       
                                1362                                      
Gly Gly Gly Asp Met Arg Asp Asn Trp Arg Ser Glu Leu                       
            445                 450                                       
TAT AAA TAT AAA GTA GTA AAA GTT GAA CCA TTA GGA ATA                       
                                1401                                      
Tyr Lys Tyr Lys Val Val Lys Val Glu Pro Leu Gly Ile                       
455                 460                 465                               
GCA CCC ACC AAG GCA AAG AGA AGA GTG GTG CAC AGA GAC                       
                                1440                                      
Ala Pro Thr Lys Ala Lys Arg Arg Val Val His Arg Asp                       
        470                 475                 480                       
AAA AGA GCA GCA CTA GGA GCC TTG TTC CTT GGG TTC TTA                       
                                1479                                      
Lys Arg Ala Ala Leu Gly Ala Leu Phe Leu Gly Phe Leu                       
                485                 490                                   
GGA GCA TAA AAG CTT CTA GA 1499                                           
Gly Ala Xaa Lys Leu Leu                                                   
    495             499                                                   
CLONE C11.7                                                               
    GAG GTA CCT GTA TGG AAA GAA GCA ACC ACT ACT CTA                       
                                36                                        
    Glu Val Pro Val Trp Lys Glu Ala Thr Thr Thr Leu                       
      1               5                  10                               
TTT TGT GCA TCA GAT GCT AAA GCA TAT GAC ACA GAG GTG                       
                                75                                        
Phe Cys Ala Ser Asp Ala Lys Ala Tyr Asp Thr Glu Val                       
         15                  20                  25                       
CAT AAT GTT TGG GCC ACA CAT GCC TGT GTA CCC ACA GAC                       
                                114                                       
His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp                       
                 30                  35                                   
CCC AAC CCA CAA GAA ATA GAA TTG GTA AAT GTG ACA GAA                       
                                153                                       
Pro Asn Pro Gln Glu Ile Glu Leu Val Asn Val Thr Glu                       
     40                  45                  50                           
GAT TTT AAC ATG TGG AAA AAT AAA ATG GTA GAC CAG ATG                       
                                192                                       
Asp Phe Asn Met Trp Lys Asn Lys Met Val Asp Gln Met                       
             55                  60                                       
CAT GAG GAT ATA ATC AGT TTA TGG GAT GAA AGC CTA AAG                       
                                231                                       
His Glu Asp Ile Ile Ser Leu Trp Asp Glu Ser Leu Lys                       
 65                  70                  75                               
CCA TGT GTA AAG TTA ACC CCA CTT TGT GTT ACT CTA AAC                       
                                270                                       
Pro Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu Asn                       
         80                  85                  90                       
TGC AGT GAT GTG AAC AAT TCC ACA AAT CCT AAT GAT ACT                       
                                309                                       
Cys Ser Asp Val Asn Asn Ser Thr Asn Pro Asn Asp Thr                       
                 95                 100                                   
AAT ACT AAT TCC ACT AAT ACT ACT TCC TCT ACT CCT ACG                       
                                348                                       
Asn Thr Asn Ser Thr Asn Thr Thr Ser Ser Thr Pro Thr                       
    105                 110                 115                           
GCC ACT ACT AGT AGC GAG GAA AAG ATG GAG AAG GGA GAA                       
                                387                                       
Ala Thr Thr Ser Ser Glu Glu Lys Met Glu Lys Gly Glu                       
            120                 125                                       
ATA AAA AAC TGC TCT TTC AAT ATC ACC ACA CAC ATG AAA                       
                                426                                       
Ile Lys Asn Cys Ser Phe Asn Ile Thr Thr His Met Lys                       
130                 135                 140                               
GAT AAG GTA CAG AAA GAA TAT GCA CTT TTT TAT AAA CTT                       
                                465                                       
Asp Lys Val Glfl Lys Glu Tyr Ala Leu Phe Tyr Lys Leu                      
        145                 150                 155                       
GAT ATA GTA CCA ATA GAT GAT AAT AAT ACC AGC TAT AGG                       
                                504                                       
Asp Ile Val Pro Ile Asp Asp Asn Asn Thr Ser Tyr Arg                       
                160                 165                                   
TTG ATA AGT TGT AAT ACC TCA GTC ATT ACA CAG GCC TGT                       
                                543                                       
Leu Ile Ser Cys Asn Thr Ser Val Ile Thr Gln Ala Cys                       
    170                 175                 180                           
CCA ATG GTG ACC TTT GAG CCA ATT CCC ATA CAT TAT TGT                       
                                582                                       
Pro Met Val Thr Phe Glu Pro Ile Pro Ile His Tyr Cys                       
            185                 190                                       
GCC CCG GCT GGT TTT GCG ATT CTA AAG TGT AAA GAT AAG                       
                                621                                       
Ala Pro Ala Gly Phe Ala Ile Leu Lys Cys Lys Asp Lys                       
195                 200                 205                               
AAG TTC AAT GGA ACA GGA CCA TGT TCA AAG GTC AGC ACA                       
                                660                                       
Lys Phe Asn Gly Thr Gly Pro Cys Ser Lys Val Ser Thr                       
        210                 215                 220                       
GTA CAA TGT ACA CAT GGA ATT AGG CCA GTA GTA TCA ACT                       
                                699                                       
Val Gln Cys Thr His Gly Ile Arg Pro Val Val Ser Thr                       
                225                 230                                   
CAA CTG TTG TTA AAT GGC AGT CTT GCA GAA GAA GAA GTA                       
                                738                                       
Gln Leu Leu Leu Asn Gly Ser Leu Ala Glu Glu Glu Val                       
    235                 240                 245                           
GTA ATT AGA TCT GTC AAT TTC ACA GAC AAT GCT AAA ATC                       
                                777                                       
Val Ile Arg Ser Val Asn Phe Thr Asp Asn Ala Lys Ile                       
            250                 255                                       
ATA ATA GTA CAG CTG AAA GAA CCT GTA GCA ATT AAT TGT                       
                                816                                       
Ile Ile Val Gln Leu Lys Glu Pro Val Ala Ile Asn Cys                       
260                 265                 270                               
ACA AGA CCC AAC AAC AAT ACA AGA AAA GGT ATA CAT CTA                       
                                855                                       
Thr Arg Pro Asn Asn Asn Thr Arg Lys Gly Ile His Leu                       
        275                 280                 285                       
GGA CCA GGG AGC ACA TTT TAT ACA ACA GGA GAA ATA ATA                       
                                894                                       
Gly Pro Gly Ser Thr Phe Tyr Thr Thr Gly Glu Ile Ile                       
                290                 295                                   
GGA GAC ATA AGA AAA GCA TAT TGC AAG ATT AGT AAA GAA                       
                                933                                       
Gly Asp Ile Arg Lys Ala Tyr Cys Lys Ile Ser Lys Glu                       
    300                 305                 310                           
AAA TGG AAT AAC ACT TTA AGA CAG GTA GTT AAA AAA TTA                       
                                972                                       
Lys Trp Asn Asn Thr Leu Arg Gln Val Val Lys Lys Leu                       
            315                 320                                       
AGA GAA CAA TTT GGG AAT AAA ACA ATA ATT TTT AAT CGA                       
                                1011                                      
Arg Glu Gln Phe Gly Asn Lys Thr Ile Ile Phe Asn Arg                       
325                 330                 335                               
TCC TCA GGA GGG GAC CCA GAA ATT GTA ATG CAC AGT TTT                       
                                1050                                      
Ser Ser Gly Gly Asp Pro Glu Ile Val Met His Ser Phe                       
        340                 345                 350                       
AAC TGT GGA GGG GAG TTT TTC TAC TGT AAT ACA ACA CAA                       
                                1089                                      
Asn Cys Gly Gly Glu Phe Phe Tyr Cys Asn Thr Thr Gln                       
                355                 360                                   
CTG TTT AAT AGT ACT TGG AAT AAT ACT GAA GGG ACA AAT                       
                                1128                                      
Leu Phe Asn Ser Thr Trp Asn Asn Thr Glu Gly Thr Asn                       
    365                 370                 375                           
AGC ACT GAA GGA AAT AGC ACA ATC ACA CTC CCA TGC AGA                       
                                1167                                      
Ser Thr Glu Gly Asn Ser Thr Ile Thr Leu Pro Cys Arg                       
            380                 385                                       
ATA AAA CAA ATT ATA AAT ATG TGG CAG GAA GTA GGA AAA                       
                                1206                                      
Ile Lys Gln Ile Ile Asn Met Trp Gln Glu Val Gly Lys                       
390                 395                 400                               
GCA ACG TAT GCC CCT CCC ATC AGA GGA CGA ATT AGA TGC                       
                                1245                                      
Ala Thr Tyr Ala Pro Pro Ile Arg Gly Arg Ile Arg Cys                       
        405                 410                 415                       
ATA TCA AAT ATT ACA GGA CTG CTA TTA ACA AGA GAT GGT                       
                                1284                                      
Ile Ser Asn Ile Thr Gly Leu Leu Leu Thr Arg Asp Gly                       
                420                 425                                   
GGT AGG AAT GTC ACA AAC AAT ACC GAN NCC TTC AGA CCT                       
                                1323                                      
Gly Arg Asn Val Thr Asn Asn Thr Xaa Xaa Phe Arg Pro                       
    430                 435                 440                           
GGA GGA GGA GAC ATG AGG GAC AAT TGG AGA AGT GAA TTA                       
                                1362                                      
Gly Gly Gly Asp Met Arg Asp Asn Trp Arg Ser Glu Leu                       
            445                 450                                       
TAT AAA TAT AAA GTA GTA AAA GTT GAA CCA TTA GGA ATA                       
                                1401                                      
Tyr Lys Tyr Lys Val Val Lys Val Glu Pro Leu Gly Ile                       
455                 460                 465                               
GCA CCC ACC AAG GCA AAG AGA AGA GTG GTG CAC AGA GAC                       
                                1440                                      
Ala Pro Thr Lys Ala Lys Arg Arg Val Val His Arg Asp                       
        470                 475                 480                       
AAA AGA GCA GCA CTA GGA GCT TTG TTC CTT GGG TTC TTA                       
                                1479                                      
Lys Arg Ala Ala Leu Gly Ala Leu Phe Leu Gly Phe Leu                       
                485                 490                                   
GGA GCA TAA AAG CTT CTA GA 1499                                           
Gly Ala Xaa Lys Leu Leu                                                   
     495             499                                                  
CLONE C10.5                                                               
G   GTA CCT GTG TGG AAA GAA GCA AAC ACA ACT CTA TTT                       
                                37                                        
    Val Pro Val Trp Lys Glu Ala Asn Thr Thr Leu Phe                       
      1               5                  10                               
TGT GCA TCA GAT GCT AAA GCA TAT GAT AGA GAA GTA CAT                       
                                76                                        
Cys Ala Ser Asp Ala Lys Ala Tyr Asp Arg Glu Val His                       
          15                  20                  25                      
AAT GTT TGG GCA ACA CAT GCC TGT GTA CCC ACA GAC CCC                       
                                115                                       
Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro                       
                 30                  35                                   
AAC CCA CAA GAA ATA GTA TTG GGA AAT GTG ACA GAA AAT                       
                                154                                       
Asn Pro Gln Glu Ile Val Leu Gly Asn Val Thr Glu Asn                       
     40                  45                  50                           
TTT AAC ATG TGG AAA AAT AAC ATG GTA GAA CAA ATG CAT                       
                                193                                       
Phe Asn Met Trp Lys Asn Asn Met Val Glu Gln Met His                       
             55                  60                                       
GAG GAT ATA ATC AAT TTA TGG GAT CAA AGC TTA AAG CCA                       
                                232                                       
Glu Asp Ile Ile Asn Leu Trp Asp Gln Ser Leu Lys Pro                       
 65                  70                  75                               
TGT GTA AAG TTA ACT CCA CTC TGT GTT ACT TTA AAG TGC                       
                                271                                       
Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu Lys Cys                       
         80                  85                  90                       
AAG GAT CTG GAG AGG AAT ACT ACC TAT AAT AGC ACT ATT                       
                                310                                       
Lys Asp Leu Glu Arg Asn Thr Thr Tyr Asn Ser Thr Ile                       
                 95                 100                                   
ACC AAT AAT AGT AGT TTG GAG GGA CTA AGA GAA CAA ATG                       
                                349                                       
Thr Asn Asn Ser Ser Leu Glu Gly Leu Arg Glu Gln Met                       
    105                 110                 115                           
ACA AAC TGC TCT TTC AAC ATC ACC ACA AGT ATA AGA GAT                       
                                388                                       
Thr Asn Cys Ser Phe Asn Ile Thr Thr Ser Ile Arg Asp                       
            120                 125                                       
AAG GTG CAG AAA GAA TAT GCA CTT TTG TAT AAA CTT GAT                       
                                427                                       
Lys Val Gln Lys Glu Tyr Ala Leu Leu Tyr Lys Leu Asp                       
130                 135                 140                               
GTA GTA CCA ATA GAA GAA GAT GAC AAT ACT AGC TAT AGA                       
                                466                                       
Val Val Pro Ile Glu Glu Asp Asp Asn Thr Ser Tyr Arg                       
        145             150                     155                       
TTG ATA AGT TGT AAC ACC TCA GTC ATT ACA CAG GCT TGT                       
                                505                                       
Leu Ile Ser Cys Asn Thr Ser Val Ile Thr Gln Ala Cys                       
                160                 165                                   
CCA AAG ACA TCC TTT GAG CCA ATT CCC ATA CAT TAT TGT                       
                                544                                       
Pro Lys Thr Ser Phe Glu Pro Ile Pro Ile His Tyr Cys                       
    170                 175                 180                           
GCC CCG GCT GGT TTT GCG ATT CTA AAG TGT AAT GAT AAG                       
                                583                                       
Ala Pro Ala Gly Phe Ala Ile Leu Lys Cys Asn Asp Lys                       
            185                 190                                       
AAG TTC AAT GGA ACA GGA CCA TGT AAA AAT GTC AGC ACA                       
                                622                                       
Lys Phe Asn Gly Thr Gly Pro Cys Lys Asn Val Ser Thr                       
195                 200                 205                               
GTA CAA TGT ACA CAT GGA ATT AGG CCA GTA GTA TCA ACT                       
                                661                                       
Val Gln Cys Thr His Gly Ile Arg Pro Val Val Ser Thr                       
        210                 215                 220                       
CAA CTG TTG TTA AAT GGC AGT CTA GCA GAA GAA GAG GTA                       
                                700                                       
Gln Leu Leu Leu Asn Gly Ser Leu Ala Glu Glu Glu Val                       
                225                 230                                   
GTA ATC AGA TCT GCC AAT TTC ACA GAC AAT GCT AAA ACC                       
                                739                                       
Val Ile Arg Ser Ala Asn Phe Thr Asp Asn Ala Lys Thr                       
    235                 240                 245                           
ATA ATA GTA CAT CTA AAT GAA ACT GTA AAA ATT AAT TGT                       
                                778                                       
Ile Ile Val His Leu Asn Glu Thr Val Lys Ile Asn Cys                       
            250                 255                                       
ACA AGA CTT GGC AAC AAT ACA AGA AAA AGT ATA AAT ATA                       
                                817                                       
Thr Arg Leu Gly Asn Asn Thr Arg Lys Ser Ile Asn Ile                       
260                 265                 270                               
GGA CCA GGG AGA GTA CTC TAT GCA ACA GGA GAA ATA ATA                       
                                856                                       
Gly Pro Gly Arg Val Leu Tyr Ala Thr Gly Glu Ile Ile                       
        275                 280                 285                       
GGA GAC ATA AGA CAA GCA CAT TGT AAC ATT AGT AGA GCA                       
                                895                                       
Gly Asp Ile Arg Gln Ala His Cys Asn Ile Ser Arg Ala                       
                290                 295                                   
CAA TGG AAT AAG ACT TTA GAA AAG GTA GTT GAC AAA TTA                       
                                934                                       
Gln Trp Asn Lys Thr Leu Glu Lys Val Val Asp Lys Leu                       
    300                 305                 310                           
AGA AAA CAA TTT GGG GAT AAT ACA ACA ATA GCT TTT AAT                       
                                973                                       
Arg Lys Gln Phe Gly Asp Asn Thr Thr Ile Ala Phe Asn                       
            315                 320                                       
CGA TCC TCA GGA GGG GAC CCA GAA ATT GTA ATG CAC ACT                       
                                1012                                      
Arg Ser Ser Gly Gly Asp Pro Glu Ile Val Met His Thr                       
325                 330                 335                               
TTT AAT TGT GGA GGG GAA TTT TTC TAC TGT AAT ACA ACA                       
                                1051                                      
Phe Asn Cys Gly Gly Glu Phe Phe Tyr Cys Asn Thr Thr                       
        340                 345                 350                       
CAA CTG TTT AAT AGT ACT TGG AAT AAT ACT TGG AAG GAT                       
                                1090                                      
Gln Leu Phe Asn Ser Thr Trp Asn Asn Thr Trp Lys Asp                       
                355                 360                                   
CCT AAC AGG AGT GAC AAT ATC ACA CTC CCA TGC AGA ATA                       
                                1129                                      
Pro Asn Arg Ser Asp Asn Ile Thr Leu Pro Cys Arg Ile                       
    365                 370                 375                           
AAA CAA ATT ATA AAC ATG TGG CAG GAA GTA GGA AAA GCA                       
                                1168                                      
Lys Gln Ile Ile Asn Met Trp Gln Glu Val Gly Lys Ala                       
            380                 385                                       
ATG TAC GCC CCT CCC ATC AGA GGG GAA ATT AGA TGT TCA                       
                                1207                                      
Met Tyr Ala Pro Pro Ile Arg Gly Glu Ile Arg Cys Ser                       
390                 395               400                                 
TCA AAT ATC ACA GGG CTG CTA CTA ACA AGA GAT GGT GGT                       
                                1246                                      
Ser Asn Ile Thr Gly Leu Leu Leu Thr Arg Asp Gly Gly                       
        405                 410                 415                       
AAT GAC GAT GGT AAT GAC ACG ACC ACA AAC AGG ACC GAG                       
                                1285                                      
Asn Asp Asp Gly Asn Asp Thr Thr Thr Asn Arg Thr Glu                       
                420                 425                                   
ATC TTC AGA CCT GGA GGA GGA GAT ATG AGG GAC AAT TGG                       
                                1324                                      
Ile Phe Arg Pro Gly Gly Gly Asp Met Arg Asp Asn Trp                       
    430                 435                 440                           
AGA AGT GAA TTA TAT AGA TAT AAA GTA GTA AAA ATT GAA                       
                                1363                                      
Arg Ser Glu Leu Tyr Arg Tyr Lys Val Val Lys Ile Glu                       
            445                 450                                       
CCA TTA GGA ATA GCA CCC ACC AGG GCA AAG AGA AGA GTG                       
                                1402                                      
Pro Leu Gly Ile Ala Pro Thr Arg Ala Lys Arg Arg Val                       
455                 460                 465                               
GTG CAG AGA GAA AAA AGA GCA GTA GGA CTA GGA GCT TTG                       
                                1441                                      
Val Gln Arg Glu Lys Arg Ala Val Gly Leu Gly Ala Leu                       
        470                 475                 480                       
TTC CTT GGG T TCTTAGGAG CATAAAGCTT CTAGA 1475                             
Phe Leu Gly                                                               
        483                                                               
CLONE C10.7                                                               
G   GTA CCT GTG TGG AAA GAA GCA AAC ACA ACT CTA TTT                       
                                37                                        
    Val Pro Val Trp Lys Glu Ala Asn Thr Thr Leu Phe                       
      1               5                  10                               
TGT GCA TCA GAT GCT AAA GCA TAT GAT AGA GAA GTA CAT                       
                                76                                        
Cys Ala Ser Asp Ala Lys Ala Tyr Asp Arg Glu Val His                       
         15                  20                  25                       
AAT GTT TGG GCA ACA CAT GCC TGT GTA CCC ACA GAC CCC                       
                                115                                       
Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro                       
                 30                  35                                   
AAC CCA CAA GAA ATA GTA TTG GGA AAT GTG ACA GAA AAT                       
                                154                                       
Asn Pro Gln Glu Ile Val Leu Gly Asn Val Thr Glu Asn                       
     40                  45                  50                           
TTT AAC ATG TGG AAA AAT AAC ATG GTA GAA CAA ATG CAT                       
                                193                                       
Phe Asn Met Trp Lys Asn Asn Met Val Glu Gln Met His                       
             55                  60                                       
GAG GAT ATA ATC AAT TTA TGG GAT CAA AGC TTA AAG CCA                       
                                232                                       
Glu Asp Ile Ile Asn Leu Trp Asp Gln Ser Leu Lys Pro                       
 65                  70                  75                               
TGT GTA AAG TTA ACT CCA CTC TGT GTT ACT TTA AAG TGC                       
                                271                                       
Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu Lys Cys                       
         80                  85                  90                       
AAG GAT CTG GAG AGG AAT ACT ACC TAT AAT AGC ACT ATT                       
                                310                                       
Lys Asp Leu Glu Arg Asn Thr Thr Tyr Asn Ser Thr Ile                       
                 95                 100                                   
ACC AAT AAT AGT AGT TTG GAG GGA CTA AGA GAA CAA ATG                       
                                349                                       
Thr Asn Asn Ser Ser Leu Glu Gly Leu Arg Glu Gln Met                       
    105                 110                 115                           
ACA AAC TGC TCT TTC AAC ATC ACC ACA AGT ATA AGA GAT                       
                                388                                       
Thr Asn Cys Ser Phe Asn Ile Thr Thr Ser Ile Arg Asp                       
            120                 125                                       
AAG GTG CAG AAA GAA TAT GCA CTT TTG TAT AAA CTT GAT                       
                                427                                       
Lys Val Gln Lys Glu Tyr Ala Leu Leu Tyr Lys Leu Asp                       
130                 135                 140                               
GTA GTA CCA ATA GAA GAA GAT GAC AAT ACT AGC TAT AGA                       
                                466                                       
Val Val Pro Ile Glu Glu Asp Asp Asn Thr Ser Tyr Arg                       
        145                 150                 155                       
TTG ATA AGT TGT AAC ACC TCA GTC ATT ACA CAG GCT TGT                       
                                505                                       
Leu Ile Ser Cys Asn Thr Ser Val Ile Thr Gln Ala Cys                       
                160                 165                                   
CCA AAG ACA TCC TTT GAG CCA ATT CCC ATA CAT TAT TGT                       
                                544                                       
Pro Lys Thr Ser Phe Glu Pro Ile Pro Ile His Tyr Cys                       
    170                 175                 180                           
GCC CCG GCT GGT TTT GCG ATT CTA AAG TGT AAT GAT AAG                       
                                583                                       
Ala Pro Ala Gly Phe Ala Ile Leu Lys Cys Asn Asp Lys                       
            185                 190                                       
AAG TTC AAT GGA ACA GGA CCA TGT AAA AAT GTC AGC ACA                       
                                622                                       
Lys Phe Asn Gly Thr Gly Pro Cys Lys Asn Val Ser Thr                       
195                 200                 205                               
GTA CAA TGT ACA CAT GGA ATT AGG CCA GTA GTA TCA ACT                       
                                661                                       
Val Gln Cys Thr His Gly Ile Arg Pro Val Val Ser Thr                       
        210                 215                 220                       
CAA CTG TTG TTA AAT GGC AGT CTA GCA GAA GAA GAG GTA                       
                                700                                       
Gln Leu Leu Leu Asn Gly Ser Leu Ala Glu Glu Glu Val                       
                225                 230                                   
GTA ATC AGA TCT GCC AAT TTC ACA GAC AAT GCT AAA ACC                       
                                739                                       
Val Ile Arg Ser Ala Asn Phe Thr Asp Asn Ala Lys Thr                       
     235                240                 245                           
ATA ATA GTA CAT CTA AAT GAA ACT GTA AAA ATT AAT TGT                       
                                778                                       
Ile Ile Val His Leu Asn Glu Thr Val Lys Ile Asn Cys                       
            250                 255                                       
ACA AGA CTT GGC AAC AAT ACA AGA AAA AGT ATA AAT ATA                       
                                817                                       
Thr Arg Leu Gly Asn Asn Thr Arg Lys Ser Ile Asn Ile                       
260                 265                 270                               
GGA CCA GGG AGA GTA CTC TAT GCA ACA GGA GAA ATA ATA                       
                                856                                       
Gly Pro Gly Arg Val Leu Tyr Ala Thr Gly Glu Ile Ile                       
        275                 280                 285                       
GGA GAC ATA AGA CAA GCA CAT TGT AAC ATT AGT AGA GCA                       
                                895                                       
Gly Asp Ile Arg Gln Ala His Cys Asn Ile Ser Arg Ala                       
                290                 295                                   
CAA TGG AAT AAG ACT TTA GAA AAG GTA GTT GAC AAG TTA                       
                                934                                       
Gln Trp Asn Lys Thr Leu Glu Lys Val Val Asp Lys Leu                       
    300                 305                 310                           
AGA AAA CAA TTT GGG GAT AAT ACA ACA ATA GCT TTT AAT                       
                                973                                       
Arg Lys Gln Phe Gly Asp Asn Thr Thr Ile Ala Phe Asn                       
            315                 320                                       
CGA TCC TCA GGA GGG GAC CCA GAA ATT GTA ATG CAC ACT                       
                                1012                                      
Arg Ser Ser Gly Gly Asp Pro Glu Ile Val Met His Thr                       
325                 330                 335                               
TTT AAT TGT GGA GGG GAA TTT TTC TAC TGT AAT ACA ACA                       
                                1051                                      
Phe Asn Cys Gly Gly Glu Phe Phe Tyr Cys Asn Thr Thr                       
        340                 345                 350                       
CAA CTG TTT AAT AGT ACT TGG AAT AAT ACT TGG AAG GAT                       
                                1090                                      
Gln Leu Phe Asn Ser Thr Trp Asn Asn Thr Trp Lys Asp                       
                355                 360                                   
CCT AAC AGG AGT GAC AAT ATC ACA CTC CCA TGC AGA ATA                       
                                1129                                      
Pro Asn Arg Ser Asp Asn Ile Thr Leu Pro Cys Arg Ile                       
    365                 370                 375                           
AAA CAA ATT ATA AAC ATG TGG CAG GAA GTA GGA AAA GCA                       
                                1168                                      
Lys Gln Ile Ile Asn Met Trp Gln Glu Val Gly Lys Ala                       
            380                 385                                       
ATG TAC GCC CCT CCC ATC AGA GGG GAA ATT AGA TGT TCA                       
                                1207                                      
Met Tyr Ala Pro Pro Ile Arg Gly Glu Ile Arg Cys Ser                       
390                 395                 400                               
TCA AAT ATC ACA GGG CTG CTA CTA ACA AGA GAT GGT GGT                       
                                1246                                      
Ser Asn Ile Thr Gly Leu Leu Leu Thr Arg Asp Gly Gly                       
        405                 410                 415                       
AAT GAC GAT GGT AAT GAC ACG ACC ACA AAC AGG ACC GAG                       
                                1285                                      
Asn Asp Asp Gly Asn Asp Thr Thr Thr Asn Arg Thr Glu                       
                420                 425                                   
ATC TTC AGA CCT GGA GGA GGA GAT ATG AGG GAC AAT TGG                       
                                1324                                      
Ile Phe Arg Pro Gly Gly Gly Asp Met Arg Asp Asn Trp                       
     430                435                 440                           
AGA AGT GAA TTA TAT AGA TAT AAA GTA GTA AAA ATT GAA                       
                                1363                                      
Arg Ser Glu Leu Tyr Arg Tyr Lys Val Val Lys Ile Glu                       
            445                 450                                       
CCA TTA GGA ATA GCA CCC ACC AGG GCA AAG AGA AGA GTG                       
                                1402                                      
Pro Leu Gly Ile Ala Pro Thr Arg Ala Lys Arg Arg Val                       
455                 460                 465                               
GTG CAG AGA GAA AAA AGA GCA GTA GGA CTA GGA GCT TTG                       
                                1441                                      
Val Gln Arg Glu Lys Arg Ala Val Gly Leu Gly Ala Leu                       
        470                 475                 480                       
TTC CTT GGG TTC TTG GGA GCA TAA AGC TTC TAG A 1475                        
Phe Leu Gly Phe Leu Gly Ala Xaa Ser Phe Xaa                               
                485                 490 491                               
CLONE C17.1                                                               
    CTC GAG GTA CCT GTG TGG AAA GAA GCA ACC ACC ACT                       
                                36                                        
    Leu Glu Val Pro Val Trp Lys Glu Ala Thr Thr Thr                       
      1               5                  10                               
CTA TTT TGT GCA TCA GAT GCT AAA GCA TAT GAT TCA GAG                       
                                75                                        
Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Asp Ser Glu                       
         15                  20                  25                       
GCA CAT AAT GTT TGG GCC ACA CAT GCC TGT GTA CCC ACA                       
                                114                                       
Ala His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr                       
                 30                  35                                   
GAC CCC AAC CCA CAA GAA GTA GAA TTG GAA AAT GTG ACA                       
                                153                                       
Asp Pro Asn Pro Gln Glu Val Glu Leu Glu Asn Val Thr                       
     40                  45                  50                           
GAA AAT TTT AAC ATG TGG AAA AAT AAC ATG GTA GAA CAG                       
                                192                                       
Glu Asn Phe Asn Met Trp Lys Asn Asn Met Val Glu Gln                       
             55                  60                                       
ATG CAT GGG GAT ATA ATT AGT TTA TGG GAT CAA AGC CTA                       
                                231                                       
Met His Gly Asp Ile Ile Ser Leu Trp Asp Gln Ser Leu                       
 65                  70                  75                               
AAG CCA TGT GTA AAA TTA ACC CCA CTC TGT GTT ACG TTA                       
                                270                                       
Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu                       
         80                  85                  90                       
AAT TGC ACT GAC CCA AAT GTT ACT AAT AGC GAG AGA ACG                       
                                309                                       
Asn Cys Thr Asp Pro Asn Val Thr Asn Ser Glu Arg Thr                       
                 95                 100                                   
ATA GAG GGG GGA GAA ATA AAA AAT TGC TCT TTC AAT ATC                       
                                348                                       
Ile Glu Gly Gly Glu Ile Lys Asn Cys Ser Phe Asn Ile                       
    105                 110                 115                           
ACC ACA AAC ATA AGA GAT AGG TTT CAG AAA GAA TAT GCA                       
                                387                                       
Thr Thr Asn Ile Arg Asp Arg Phe Gln Lys Glu Tyr Ala                       
            120                 125                                       
CTT TTT TAT AAA CTT GAT GTA ATA CCA TTA GGT AAT GAT                       
                                426                                       
Leu Phe Tyr Lys Leu Asp Val Ile Pro Leu Gly Asn Asp                       
130                 135                 140                               
AAT ACT AGC TAT AGG TTG ATA AGT TGT AAC ACC TCA GTC                       
                                465                                       
Asn Thr Ser Tyr Arg Leu Ile Ser Cys Asn Thr Ser Val                       
        145                 150                 155                       
ATT ACA CAG GCC TGT CCA AAG GTA TCC TTT GAG CCA ATT                       
                                504                                       
Ile Thr Gln Ala Cys Pro Lys Val Ser Phe Glu Pro Ile                       
                160                 165                                   
CCC ATA CAT TAT TGT GCC CCG GCT GGT TTT GCG ATT CTA                       
                                543                                       
Pro Ile His Tyr Cys Ala Pro Ala Gly Phe Ala Ile Leu                       
    170                 175                 180                           
AAG TGT AAA GAT AAG AAG TTC AAT GGA ACA GGA CCA TGT                       
                                582                                       
Lys Cys Lys Asp Lys Lys Phe Asn Gly Thr Gly Pro Cys                       
            185                 190                                       
ACA AAT GTC AGC ACA GTA CAA TGT ACA CAT GGA ATT AAG                       
                                621                                       
Thr Asn Val Ser Thr Val Gln Cys Thr His Gly Ile Lys                       
195                 200                 205                               
CCA GTA GTA TCA ACT CAA CTG TTG TTA AAT GGC AGT CTA                       
                                660                                       
Pro Val Val Ser Thr Gln Leu Leu Leu Asn Gly Ser Leu                       
        210                 215                 220                       
GCA GAA GAA GAC ATA GTA ATT AGA TCC GCC AAT CTC ACA                       
                                699                                       
Ala Glu Glu Asp Ile Val Ile Arg Ser Ala Asn Leu Thr                       
                225                 230                                   
GAC AAT GCT AAA AAC ATA ATA GTA CAG CTG AAT GAA TCT                       
                                738                                       
Asp Asn Ala Lys Asn Ile Ile Val Gln Leu Asn Glu Ser                       
    235                 240                 245                           
GTA ACA ATG AAT TGT ACA AGA CCC AAC AAC AAT ACA ATG                       
                                777                                       
Val Thr Met Asn Cys Thr Arg Pro Asn Asn Asn Thr Met                       
            250                 255                                       
AAA AGT ATA CAT ATA GGA CCA GGC AGA GCA TTT TAT GCA                       
                                816                                       
Lys Ser Ile His Ile Gly Pro Gly Arg Ala Phe Tyr Ala                       
260                 265                 270                               
ACA GGA AAC ATA ATA GGA GAT ATA AGA CAA GCA CAT TGT                       
                                855                                       
Thr Gly Asn Ile Ile Gly Asp Ile Arg Gln Ala His Cys                       
        275                 280                 285                       
AAC ATT AGT GGA ACA AAA TGG AAT GAC ACT TTG AAA AAG                       
                                894                                       
Asn Ile Ser Gly Thr Lys Trp Asn Asp Thr Leu Lys Lys                       
                290                 295                                   
ATA GCT ATA AAA TTA AGA GAA CAA TTT AAT AAG ACA ATA                       
                                933                                       
Ile Ala Ile Lys Leu Arg Glu Gln Phe Asn Lys Thr Ile                       
    300                 305                 310                           
GTC TTT AAT CAA TCC TCA GGA GGG GAC CCA GAA ATT GCA                       
                                972                                       
Val Phe Asn Gln Ser Ser Gly Gly Asp Pro Glu Ile Ala                       
            315                 320                                       
ACG CTC AGT TTT AAT TGT GGA GGG GAA TTT TTC TAC TGT                       
                                1011                                      
Thr Leu Ser Phe Asn Cys Gly Gly Glu Phe Phe Tyr Cys                       
325                 330                 335                               
AAT TCA ACA CAA CTG TTT AAT AGT ACT TGG AAT AGT ACT                       
                                1050                                      
Asn Ser Thr Gln Leu Phe Asn Ser Thr Trp Asn Ser Thr                       
        340                 345                 350                       
GGG TCA AAT AAC ACT AAA GGA AAT GAC ACA ATC ACA CTC                       
                                1089                                      
Gly Ser Asn Asn Thr Lys Gly Asn Asp Thr Ile Thr Leu                       
                355                 360                                   
CCA TGC AGA ATA AGA CAA ATT ATA AAC ATG TGG CAG AAA                       
                                1128                                      
Pro Cys Arg Ile Arg Gln Ile Ile Asn Met Trp Gln Lys                       
    365                 370                 375                           
ATA GGA AAA GCA ATG TAT GCC CCT CCC ATC AAA GGG CAA                       
                                1167                                      
Ile Gly Lys Ala Met Tyr Ala Pro Pro Ile Lys Gly Gln                       
            380                 385                                       
ATT AGA TGT TCA TCA AAT ATT ACA GGG CTA ATA TTA ACA                       
                                1206                                      
Ile Arg Cys Ser Ser Asn Ile Thr Gly Leu Ile Leu Thr                       
390                 395                 400                               
AGA GAT GGT GGT AAC AAC AAC ATG AGC AAG ACC ACC GAG                       
                                1245                                      
Arg Asp Gly Gly Asn Asn Asn Met Ser Lys Thr Thr Glu                       
        405                 410                 415                       
ACC TTC AGA CCT GGA GGA GGA GAT ATG AGG GAC AAT TGG                       
                                1284                                      
Thr Phe Arg Pro Gly Gly Gly Asp Met Arg Asp Asn Trp                       
                420                 425                                   
AGA AGT GAA TTA TAT AAA TAT AAA GTA GTA AAA ATT GAA                       
                                1323                                      
Arg Ser Glu Leu Tyr Lys Tyr Lys Val Val Lys Ile Glu                       
    430                 435                 440                           
CCA TTA GGA GTA GCA CCC ACC AGG GCA AAG AGA AGA GTG                       
                                1362                                      
Pro Leu Gly Val Ala Pro Thr Arg Ala Lys Arg Arg Val                       
            445                 450                                       
GTG CAG AGA GAA AAA AGA GCA GTG GGA ATA GGA GCT GTG                       
                                1401                                      
Val Gln Arg Glu Lys Arg Ala Val Gly Ile Gly Ala Val                       
455                 460                 465                               
TTC CTT GGG TTC TTG GGA GCA TAA AGC TTC TAG A 1435                        
Phe Leu Gly Phe Leu Gly Ala Xaa Ser Phe Xaa                               
        470                 475         478                               
CLONE C17.3                                                               
    CTC GAG GTA CCT GTG TGG AAA GAA GCA ACC ACC ACT                       
                                36                                        
    Leu Glu Val Pro Val Trp Lys Glu Ala Thr Thr Thr                       
      1               5                  10                               
CTA TTT TGT GCA TCA GAT GCT AAA GCA TAT GAT TCA GAG                       
                                75                                        
Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Asp Ser Glu                       
         15                  20                  25                       
GCA CAT AAT GTT TGG GCC ACA CAT GCC TGT GTA CCC ACA                       
                                114                                       
Ala His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr                       
                 30                  35                                   
GAC CCC AAC CCA CAA GAA GTA GAA TTG GAA AAT GTG ACA                       
                                153                                       
Asp Pro Asn Pro Gln Glu Val Glu Leu Glu Asn Val Thr                       
     40                  45                  50                           
GAA AAT TTT AAC ATG TGG AAA AAT AAC ATG GTA GAA CAG                       
                                192                                       
Glu Asn Phe Asn Met Trp Lys Asn Asn Met Val Glu Gln                       
            55                   60                                       
ATG CAT GGG GAT ATA ATT AGT TTA TGG GAT CAA AGC CTA                       
                                231                                       
Met His Gly Asp Ile Ile Ser Leu Trp Asp Gln Ser Leu                       
 65                  70                  75                               
AAG CCA TGT GTA AAA TTA ACC CCA CTC TGT GTT ACG TTA                       
                                270                                       
Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu                       
         80                  85                  90                       
AAT TGC ACT GAC CCA AAT GTT ACT AAT AGC GAG AGA ACG                       
                                309                                       
Asn Cys Thr Asp Pro Asn Val Thr Asn Ser Glu Arg Thr                       
                 95                 100                                   
ATA GAG GGG GGA GAA ATA AAA AAT TGC TCT TTC AAT ATC                       
                                348                                       
Ile Glu Gly Gly Glu Ile Lys Asn Cys Ser Phe Asn Ile                       
    105                 110                 115                           
ACC ACA AAC ATA AGA GAT AGG TTT CAG AAA GAA TAT GCA                       
                                387                                       
Thr Thr Asn Ile Arg Asp Arg Phe Gln Lys Glu Tyr Ala                       
            120                 125                                       
CTT TTT TAT AAA CTT GAT GTA ATA CCA TTA GGT AAT GAT                       
                                426                                       
Leu Phe Tyr Lys Leu Asp Val Ile Pro Leu Gly Asn Asp                       
130                 135                 140                               
AAT ACT AGC TAT AGG TTG ATA AGT TGT AAC ACC TCA GTC                       
                                465                                       
Asn Thr Ser Tyr Arg Leu Ile Ser Cys Asn Thr Ser Val                       
        145                 150                 155                       
ATT ACA CAG GCC TGT CCA AAG GTA TCC TTT GAG CCA ATT                       
                                504                                       
Ile Thr Gln Ala Cys Pro Lys Val Ser Phe Glu Pro Ile                       
                160                 165                                   
CCC ATA CAT TAT TGT GCC CCG GCT GGT TTT GCG ATT CTA                       
                                543                                       
Pro Ile His Tyr Cys Ala Pro Ala Gly Phe Ala Ile Leu                       
    170                 175                 180                           
AAG TGT AAA GAT AAG AAG TTC AAT GGA ACA GGA CCA TGT                       
                                582                                       
Lys Cys Lys Asp Lys Lys Phe Asn Gly Thr Gly Pro Cys                       
            185                 190                                       
ACA AAT GTC AGC ACA GTA CAA TGT ACA CAT GGA ATT AAG                       
                                621                                       
Thr Asn Val Ser Thr Val Gln Cys Thr His Gly Ile Lys                       
195                 200                 205                               
CCA GTA GTA TCA ACT CAA CTG TTG TTA AAT GGC AGT CTA                       
                                660                                       
Pro Val Val Ser Thr Gln Leu Leu Leu Asn Gly Ser Leu                       
        210                 215                 220                       
GCA GAA GAA GAC ATA GTA ATT AGA TCC GCC AAT CTC ACA                       
                                699                                       
Ala Glu Glu Asp Ile Val Ile Arg Ser Ala Asn Leu Thr                       
                225                 230                                   
GAC AAT GCT AAA AAC ATA ATA GTA CAG CTG AAT GAA TCT                       
                                738                                       
Asp Asn Ala Lys Asn Ile Ile Val Gln Leu Asn Glu Ser                       
    235                 240                 245                           
GTA ACA ATG AAT TGT ACA AGA CCC AAC AAC AAT ACA ATG                       
                                777                                       
Val Thr Met Asn Cys Thr Arg Pro Asn Asn Asn Thr Met                       
            250                 255                                       
AAA AGT ATA CAT ATA GGA CCA GGC AGA GCA TTT TAT GCA                       
                                816                                       
Lys Ser Ile His Ile Gly Pro Gly Arg Ala Phe Tyr Ala                       
260                 265                 270                               
ACA GGA AAC ATA ATA GGA GAT ATA AGA CAA GCA CAT TGT                       
                                855                                       
Thr Gly Asn Ile Ile Gly Asp Ile Arg Gln Ala His Cys                       
        275                 280                 285                       
AAC ATT AGT GGA ACA AAA TGG AAT GAC ACT TTG AAA AAG                       
                                894                                       
Asn Ile Ser Gly Thr Lys Trp Asn Asp Thr Leu Lys Lys                       
                290                 295                                   
ATA GCT ATA AAA TTA AGA GAA CAA TTT AAT AAG ACA ATA                       
                                933                                       
Ile Ala Ile Lys Leu Arg Glu Gln Phe Asn Lys Thr Ile                       
    300                 305                 310                           
GTC TTT AAT CAA TCC TCA GGA GGG GAC CCA GAA ATT GCA                       
                                972                                       
Val Phe Asn Gln Ser Ser Gly Gly Asp Pro Glu Ile Ala                       
            315                 320                                       
ACG CTC AGT TTT AAT TGT GGA GGG GAA TTT TTC TAC TGT                       
                                1011                                      
Thr Leu Ser Phe Asn Cys Gly Gly Glu Phe Phe Tyr Cys                       
325                 330                 335                               
AAT TCA ACA CAA CTG TTT AAT AGT ACT TGG AAT AGT ACT                       
                                1050                                      
Asn Ser Thr Gln Leu Phe Asn Ser Thr Trp Asn Ser Thr                       
        340                 345                 350                       
GGG TCA AAT AAC ACT AAA GGA AAT GAC ACA ATC ACA CTC                       
                                1089                                      
Gly Ser Asn Asn Thr Lys Gly Asn Asp Thr Ile Thr Leu                       
                355                 360                                   
CCA TGC AGA ATA AGA CAA ATT ATA AAC ATG TGG CAG AAA                       
                                1128                                      
Pro Cys Arg Ile Arg Gln Ile Ile Asn Met Trp Gln Lys                       
    365                 370                 375                           
ATA GGA AAA GCA ATG TAT GCC CCT CCC ATC AAA GGG CAA                       
                                1167                                      
Ile Gly Lys Ala Met Tyr Ala Pro Pro Ile Lys Gly Gln                       
            380                 385                                       
ATT AGA TGT TCA TCA AAT ATT ACA GGG CTA ATA TTA ACA                       
                                1206                                      
Ile Arg Cys Ser Ser Asn Ile Thr Gly Leu Ile Leu Thr                       
390                 395                 400                               
AGA GAT GGT GGT AAC AAC AAC ATG AGC AAG ACC ACC GAG                       
                                1245                                      
Arg Asp Gly Gly Asn Asn Asn Met Ser Lys Thr Thr Glu                       
        405                 410                 415                       
ACC TTC AGA CCT GGA GGA GGA GAT ATG AGG GAC AAT TGG                       
                                1284                                      
Thr Phe Arg Pro Gly Gly Gly Asp Met Arg Asp Asn Trp                       
                420                 425                                   
AGA AGT GAA TTA TAT AAA TAT AAA GTA GTA AAA ATT GAA                       
                                1323                                      
Arg Ser Glu Leu Tyr Lys Tyr Lys Val Val Lys Ile Glu                       
    430                 435                 440                           
CCA TTA GGA GTA GCA CCC ACC AGG GCA AAG AGA AGA GTG                       
                                1362                                      
Pro Leu Gly Val Ala Pro Thr Arg Ala Lys Arg Arg Val                       
            445                 450                                       
GTG CAG AGA GAA AAA AGA GCA GTG GGA ATA GGA GCT GTG                       
                                1401                                      
Val Gln Arg Glu Lys Arg Ala Val Gly Ile Gly Ala Val                       
455                 460                 465                               
TTC CTT GGG TTC TTG GGA GCA TAA AGC TTC TAG A 1435                        
Phe Leu Gly Phe Leu Gly Ala Xaa Ser Phe Xaa                               
        470                 475         478                               
__________________________________________________________________________
In addition to the listing in Table 1, FIG. 3 shows the alignment of the amino acid sequences of the clones of each of the seven isolates. Corresponding residues from various clones are in boxes. In the figure, the amino acid sequences are aligned against MN-rgp120 (SEQ. ID. NO:41).
In one embodiment, a gp120 polypeptide of this invention has the same amino acid sequence as the sequence of one of the breakthrough isolates. In another embodiment, the amino acid sequence is truncated, as described in detail hereinafter. In another embodiment, a gp120 polypeptide sequence of this invention contains a substitution, insertion, or deletion (alteration) of one or more amino acids in the sequence of a breakthrough isolate. Usually, with the exception of amino acids that are not present in a truncated amino acid sequence and eliminate an epitope, a gp120 polypeptide of this invention will include alterations in the amino acid sequence of a breakthrough isolate that do not alter the polypeptide's ability to induce the same neutralizing antibodies as the amino acid sequence of the isolate.
In general, substitutions in the amino acid sequence of a gp120 polypeptide of this invention are conservative substitutions, particularly for amino acid residues in the V2, V3, and C4 domains of gp120, which domains contain neutralizing epitopes. However, nonconservative substitutions, particularly in domains that do not contain neutralizing epitopes are contemplated.
Conservative substitutions replace an amino acid with an amino acid of similar size and character. For example, a hydrophobic residue or hydrophilic residue is replaced with another hydrophobic residue or hydrophilic residue, respectively. Amino acids can be divided into the following groups: positively charged residues (K, R and H); negatively charged residues (D and E); amides (N and Q); aromatics (F, Y, and W); hydrophobics (P, G, A, V, L, I, and M); and uncharged residues (S and T). Usually, residues within a group are replaced with another member of the group.
In one embodiment, critical amino acid residues in the V2, V3, and C4 domains of gp120 are identical to the corresponding residues in a breakthrough isolate sequence. Critical amino acid residues in the V2, V3, and C4 domains of gp120 are described in the experimental section. In another embodiment, all amino acid residues in the V2, V3, and C4 domains of gp120 are identical to corresponding residues in a breakthrough isolate sequence.
Oligonucleotide Encoding gp120 from Breakthrough Isolates
The present invention also provides novel oligonucleotides encoding gp120 from the breakthrough isolates which can be used to express gp120. An oligonucleotide of this invention encodes a polypeptide of this invention. The oligonucleotide can be DNA or RNA, usually DNA. Although numerous nucleotide sequences can encode the same amino acid sequence due to the degeneracy of the genetic code, conveniently, the oligonucleotides of this invention include a nucleotide sequence of a breakthrough isolate as illustrated in Table 1 (Sequence ID NOs:1, 4, 7, 9, 11, 15, 18, 22, 24, 27, 30, 32, 35, and 38). Usually, an oligonucleotide of this invention is less than about 5 kilobases (kb), preferably less than about 3 kb.
To express the encoded amino acid sequence, the oligonucleotide can be inserted into a transcription unit. The transcription unit can be inserted into a plasmid for production of cell lines, inserted into a virus (e.g.; vaccinia) or can be used directly as a DNA vaccine. Suitable transcription units for production of vaccine proteins are well known. A preferred expression vector, designated psvI6B5, is illustrated in Sequence ID NO:45. The vector includes an HSV-1 gD1 signal sequence joined to a linker sequence. The gp120 nucleotide sequence to be expressed starts with the Kpn I site of the gene. Since all gp120 or gp160 sequences contain this site, any gp120 nucleotide sequence can be analogously inserted into the vector and expressed. The vector ends with a polyA tail from SV40.
In addition to being useful to express a polypeptide sequence of this invention, the oligonucleotides of this invention can also be used in diagnostics to detect HIV isolates. For example, the oligonucleotide or a portion thereof encoding a neutralizing epitope can be used in branched chain DNA diagnostics or as a probe in in situ hybridization studies.
Vaccine Preparation
A gp120 polypeptide of this invention from a selected breakthrough isolate(s) in a suitable carrier is used to make a subunit vaccine. The polypeptide can be used alone, but is generally administered in a multivalent subunit vaccine that includes gp120 MN. In addition to one or more gp120 polypeptides of this invention, the vaccine generally includes the MN polypeptide (hereinafter, MN-rgp120). The vaccine usually includes about 3 to about 5 different gp120 polypeptides, but 30 or more different gp120 polypeptides can be used.
Preparation of gp120 polypeptides for use in a vaccine is well known and is described hereinafter. With the exception of the use of the selected HIV isolate, the gp120 subunit vaccine prepared in the method does not differ from gp120 subunit vaccines of the prior art.
As with prior art gp120 subunit vaccines, gp120 at the desired degree of purity and at a sufficient concentration to induce antibody formation is mixed with a physiologically acceptable carrier. A physiologically acceptable carrier is nontoxic to a recipient at the dosage and concentration employed in the vaccine. Generally, the vaccine is formulated for injection, usually intramuscular or subcutaneous injection. Suitable carriers for injection include sterile water, but preferably are physiologic salt solutions, such as normal saline or buffered salt solutions such as phosphate-buffered saline or ringer's lactate. The vaccine generally contains an adjuvant. Useful adjuvants include QS21 (Quillaja saponaria, commercially available from Cambridge Biotech, Worcester, Mass.), which stimulates cytotoxic T-cells, and alum (aluminum hydroxide adjuvant). Formulations with different adjuvants which enhance cellular or local immunity can also be used. In particular, immunopotentiators such as cytokines can be included in the vaccine. Examples of suitable immunopotentiating cytokines include interleukins, such as interleukin-2 (IL-2) and interleukin-12 (IL-12), and tumor necrosis factor-alpha (TNF-α).
Additional excipients that can be present in the vaccine include low molecular weight polypeptides (less than about 10 residues), proteins, amino acids, carbohydrates including glucose or dextrans, chelating agents such as EDTA, and other excipients that stabilize the protein or inhibit growth of microorganisms.
The vaccine can also contain other HIV proteins. In particular, gp41 or the extracellular portion of gp41 or HIV-1 core proteins such as P24, P17, and P55 can be present in the vaccine. Although the amino acid sequence of gp41 is more conserved than that of gp120, gp41 contains neutralizing epitopes. Preferably, any gp41 present in the vaccine is from an HIV isolate present in the vaccine. gp160 from an isolate used in the vaccine can replace gp120 in the vaccine or be used together with gp120 from the isolate. Alternatively, gp160 from a different isolate than those in the vaccine can additionally be present in the vaccine.
Vaccines according to the invention can also contain one or more soluble gp120 polypeptide sequences, or fragments thereof, in combination with an engineered virus specifically designed to express proteins that induce a cytotoxic T-cell response. Suitable engineered viruses are derived from, for example, Canary Pox virus, vaccinia viruses, attenuated human herpes viruses (such as, e.g., herpes simplex viruses), and Varicella Zoster. Exemplary engineered viruses are modified to express any HIV protein capable of inducing a cytotoxic T-cell response, such as those described above. Typically, immunization with the gp120/engineered virus vaccine is followed by administration of one or more doses of the gp120 polypeptide sequence(s) to boost the immune response. If desired, viruses can be engineered to express one or more gp120 polypeptide sequences of the invention, or fragments thereof, and used in vaccines with or without soluble gp120 polypeptide sequences.
Vaccine formulations generally include a total of about 300 to 600 μg of gp120, conveniently in about 1.0 ml of carrier. Preferred formulations include use of twice the weight of a gp120 polypeptide in twice as600 μg alum. However, formulations having smaller amounts (e.g.; 50 μg per dose) are also used, generally with alum or other adjuvants. The amount of gp120 for any isolate present in the vaccine will vary depending on the immunogenicity of the gp120. For example, gp120 from some strains of HIV may be less immunogenic than gp120 from the MN strain (Sequence ID NO:41). If two strains having different immunogenicity are used in combination, empirical titration of the amount of each virus would be performed to determine the percent of the gp120 of each strain in the vaccine. For isolates having similar immunogenicity, approximately equal amounts of each isolate's gp120 would be present in the vaccine. For example, in a preferred embodiment, the vaccine includes gp120 from the MN and a strain of this invention at concentrations of about 300 μg per strain in about 1.0 ml of carrier. When the vaccine includes gp120 from about 30 isolates, about 10 to about 50 μg can be used. Methods of determining the relative amount of an immunogenic protein in multivalent vaccines are well known and have been used, for example, to determine relative proportions of various isolates in multivalent polio vaccines.
The vaccines of this invention are administered in the same manner as prior art HIV gp120 subunit vaccines. In particular, the vaccines are generally administered at 0, 1, and at 6, 8 or 12 months, depending on the protocol. A preferred protocol includes administration at 0, 1, 6, and 12 months. Following the immunization procedure, annual or bi-annual boosts can be administered. However, during the immunization process and thereafter, neutralizing antibody levels can be assayed and the protocol adjusted accordingly.
The vaccine is administered to uninfected individuals. In addition, the vaccine can be administered to seropositive individuals to augment immune response to the virus, as with prior art HIV vaccines. It is also contemplated that DNA encoding the strains of gp120 for the vaccine can be administered in a suitable vehicle for expression in the host. In this way, gp120 can be produced in the infected host, eliminating the need for repeated immunizations. Preparation of gp120 expression vehicles is described hereinafter.
Although the gp120 isolates described herein can be used as a vaccine as described above, the amino acid sequences can also be used alone or in combinations in the same type of formulation for use as an immunogen, to induce antibodies that recognize the isolate(s) present in the immunogen. Immunogens are formulated in the same manner as vaccines and can include the same excipients, etc. Antibodies induced by the immunogens can be used in a diagnostic to detect the HIV strain in the immunogen or to affinity purify the strain.
gp120 Polypeptide Sequences and Chemokine Receptors
While CD4 is the primary cellular receptor for HIV-1, it is not sufficient for entry of HIV-1 into cells. Co-receptors required in conjunction with CD4 have been identified. These co-receptors are members of the chemokine receptor family of seven-transmembrane G-protein coupled receptors. The chemokine superfamily is subdivided into two groups based on the amino terminal cysteine spacing. The CXC chemokines are primarily involved in neutrophil-mediated inflammation, and the CC chemokines tend to be involved in chronic inflammation. At least five CC chemokine receptors, designated CC-CKR1-5 (also known in the art as CCR1-5), and at least four CXC chemokine receptors, designated CXC-CKR1-4 (also known as CXCR-1-4), have been identified.
CXC-CKR-4 (CXCR-4), which has also been called the alpha-chemokine receptor fusin, serves as an entry cofactor for T-cell-tropic HIV-1 strains. CC-CKR-5 (CC-R5), which has been called beta-chemokine receptor, together with its related family members, such as CC-CKR-2b and CC-CKR3, serve as entry cofactors for macrophage-tropic HIV-1 strains. T-cell-tropic strains can infect primary T-cells and T-cell lines, but not macrophages, whereas macrophage-tropic strains can infect macrophages and primary T-cells, but not T-cell lines. T-cell- and macrophage-tropic strains are discussed more fully in Deng et. al., Nature 381:661-666 (1996), which is hereby incorporated by reference in its entirety. Examples of T-cell-tropic strains include laboratory isolates, such as IIIB and MN. Macrophage-tropic strains include primary isolates, including but not limited to A244, GNE6, GNE8, and breakthrough viruses from vaccinees immunized with gp120-based vaccines. Dual-tropic strains can use both types of co-receptors, entering cells via CXC-CKR-4 or via one or more CC-CKR family members, preferably CC-CKR-5, CC-CKR-2b, or CC-CKR-3. While the present invention is not intended to be bound or limited by any one theory, the entry of T-cell tropic and macrophagetropic HIV-1 strains is believed to provide a unifying explanation of the differences in cell tropism between viral strains, the resistance to HIV-1 infection by many CD4-transfected nonprimate cells, and the HIV-1 infection resistance of a portion of the human population.
Accordingly, in one embodiment is a vaccine containing (1) a first gp120 polypeptide sequence, or fragment thereof, from a macrophage-tropic HIV-1 strain and/or a second gp120 polypeptide sequence, or fragment thereof, from a T-cell tropic strain, in combination with (2) a breakthrough isolate HIV gp120 polypeptide sequence, or fragment thereof, from a vaccinee vaccinated with the first and/or second HIV gp120 polypeptide sequence. Preferably, the vaccine includes at least two gp120 polypeptide sequences that bind to different chemokine receptors. In one embodiment, the vaccine includes first and second gp120 polypeptide sequences that bind to different chemokine receptors. In addition, the breakthrough isolate gp120 polypeptide sequence can bind to a different chemokine receptor than the chemokine receptor(s) bound by either or both of the first and second gp120 polypeptide sequence(s).
A preferred T-cell tropic strain is a laboratory isolate, most preferably MN. Preferred macrophagetropic viruses for use in the invention are GNE6 and GNE8, which are representative of the breakthrough viruses disclosed herein and differ from MN in that their gp120s induce the formation of antibodies that recognize the gp120 sequences (e.g., the V3 domain) involved in binding to CC chemokine receptors, such as CXC-CKR-5.
In one embodiment, HIV infection is prevented by administering one or more chemokine receptor-binding gp120 polypeptide sequences, or fragment(s) thereof containing appropriate chemokine receptor-binding domains, in a vaccine, such as those described above. Preferably, the vaccine also includes one or more CD4-binding gp120 polypeptide sequences or appropriate fragments thereof. Such vaccines induce anti-HIV antibodies that inhibit viral gp120-chemokine receptor or -CD4 binding. In addition, such gp120 polypeptides can directly inhibit HIV infection by binding to one or more co-receptors for HIV infection, such as CD4 or a chemokine receptor, thus providing a prophylactic or therapeutic effect in treating HIV infection. Preferably, gp120 polypeptide sequences useful in this regard contain the T-cell binding (TCB) domain.
Various uses of chemokine receptor-binding gp120 polypeptides are discussed below with regard to the CC chemokine receptor family. However, those skilled in the art recognize that this discussion applies equally to CXC chemokine receptors that act as cofactors in HIV infection.
The gp120 polypeptides can be used as a composition containing one or more gp120 polypeptides, as described for use as a vaccine or immunogen. The composition can be administered, prophylactically or therapeutically, to a patient at risk of infection or in need of such treatment using the dosages and routes and means of administration described herein. However, chronic administration may be preferred and dosages can be adjusted accordingly. It is noted that in vivo administration can also induce antibodies that bind viral gp120, further inhibiting virus binding to CC-CKR.
The gp120 polypeptides can also be used in screening assays to identify antagonists of CC-CKR. For example, candidate antagonists can be screened for inhibition of binding of gp120 to a CC-CKR CC-CKR receptor that is isolated and attached to a surface (e.g., plastic dish) or recombinantly or naturally expressed on the surface of a cell. Antagonists can either bind gp120 or bind receptor. Preferred candidate antagonists include gp120 compounds, small gp120 peptides (5 to 20 amino acids in length, preferably 7 to 10 amino acids in length) or peptidomimetics of gp120 that bind receptor, monoclonal antibodies that bind gp120, and small organic molecules that bind either gp120 or receptor.
The antibodies induced by the gp120 polypeptides can also be used to induce anti-idiotype antibodies that bind CC chemokines. These anti-idiotype antibodies can be screened for binding to an anti-gp120 polypeptide antibody and inhibiting gp120 from binding CC-CKR receptor. Such anti-idiotype antibodies mimic gp120 by binding to CC-CKR receptor. Such antibodies, preferably human antibodies, can be obtained in a number of ways, such as human antibodies from combinatorial libraries (e.g., Burton et al. Adv. Immunolo. (1994) 57:191-280). It is now possible to produce transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, homozygous deletion of the antibody heavy-chain joining region (JH) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice results in the production of human antibodies upon antigen challenge as described in Jakobovitis et al., Proc. Natl. Acad. Sci. USA 90: 2551 (1993); Jakobovits et al., Nature 362:255-258 (1993); Bruggermann et al., Year in Immuno. 7: 33 (1993).
Alternatively, phage display technology as described by McCafferty et al., Nature 348:552-553 (1990) can be used to produce human antibodies and antibody fragments in vitro from immunoglobulin variable (V) domain gene repertoires from unimmunized donors. According to this technique, antibody V domain genes are closed in-frame either into either a major or minor coat protein gene of a filamentous bacteriophage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties. Phage display can be performed in a variety of formats as reviewed by, for example, Johnson, et al., Current Opinion in Structural Biology 3:564-571 (1993).
Several sources of V-gene segments can be used for phage display. Clackson et al., Nature, 352: 624-628 (1991) isolated a diverse array of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. A repertoire of V genes from unimmunized human donors (or embryonic cells) can be constructed. It has been demonstrated that antibodies to a diverse array of antigens (including self-antigens) can be isolated essentially following the techniques described by Marks et al., J. Mol. Biol., 222: 581-597 (1991), or Griffith et al., EMBO J., 12: 725-734 (1993).
In a natural immune response, antibody genes accumulate mutations at a high rate (somatic hypermutation). Some of the changes introduced confer higher affinity, and B cells displaying high-affinity surface immunoglobulin are preferentially replicated and differentiated during subsequent antigen challenge. This natural process can be mimicked by employing the technique known as "chain shuffling" (Marks et al., Bio/Technol. 10:779-783 [1992]). In this method, the affinity of "primary" human antibodies obtained by phage display can be improved by sequentially replacing the heavy and light chain V region genes with repertoires of naturally occurring variants (repertoires) of V domain genes obtained from unimmunized donors. This technique allows the production of antibodies and antibody fragments with affinities in the nM range. A strategy for making very large phage antibody repertoires has been described by Waterhouse et al., Nucl. Acids Res., 21: 2265-2266 (1993).
Accordingly, antibodies that bind CC-CKR can be obtained by screening antibodies or fragments thereof expressed on the surface of bacteriophage in combinatorial libraries or in other systems as described above with a gp120 monoclonal antibody that inhibits gp120 binding to receptor.
In addition to screening antibodies with a gp-120 antibody, random or combinatorial peptide libraries can be screened with either a gp120 antibody or the gp120 compounds of the invention. Approaches are available for identifying peptide ligands from libraries that comprise large collections of peptides, ranging from 1 million to 1 billion difference sequences, which can be screened using monoclonal antibodies or target molecules. The power of this technology stems from the chemical diversity of the amino acids coupled with the large number of sequences in a library. See for example, Scott et al., Cur. Open. Biotechnol. 5(1):40-8 (1994); Kenan et al. Trends Biochem. Sci. (1994) 19(2):57-64. Accordingly, the monoclonal antibodies, preferably human monoclonals or fragments thereof, generated as discussed herein, find use in treatment by inhibiting or treating HIV infection or disease progression, as well as in screening assays to identify additional pharmaceuticals.
Production of gp120
gp120 for a vaccine can be produced by any suitable means, as with prior art HIV gp120 subunit vaccines. Recombinantly-produced or chemically synthesized gp120 is preferable to gp120 isolated directly from HIV for safety reasons. Methods for recombinant production of gp120 are described below.
Oligonucleotides encoding gp120 from breakthrough isolates and capable of expressing gp120 can be prepared by conventional means. For example, the nucleotide sequence can be synthesized. Alternatively, another HIV nucleotide sequence encoding gp120 can be used as a backbone and altered at any differing residues as by site-directed mutagenesis. Site-directed mutagenesis is described in Kunkel et al, Proc. Natl. Acad. Sci. (USA) 82:488-492 (1985) and Zoller et al, Nuc. Acids Res. 10:6487-6500 (1982) and is well known.
In a preferred embodiment, the nucleotide sequence is present in an expression construct containing DNA encoding gp120 under the transcriptional and translational control of a promoter for expression of the encoded protein. The promoter can be a eukaryotic promoter for expression in a mammalian cell. In cases where one wishes to expand the promoter or produce gp120 in a prokaryotic host, the promoter can be a prokaryotic promoter. Usually a strong promoter is employed to provide high-level transcription and expression.
The expression construct can be part of a vector capable of stable extrachromosomal maintenance in an appropriate cellular host or may be integrated into host genomes. Normally, markers are provided with the expression construct which allow for selection of a host containing the construct. The marker can be on the same or a different DNA molecule, desirably, the same DNA molecule.
The expression construct can be joined to a replication system recognized by the intended host cell. Various replication systems include viral replication systems such as those from retroviruses, simian virus, bovine papilloma virus, or the like. In addition, the construct may be joined to an amplifiable gene, e.g. the DHFR gene, so that multiple copies of the gp120 DNA can be made. Introduction of the construct into the host will vary depending on the construct and can be achieved by any convenient means. A wide variety of prokaryotic and eukaryotic hosts can be employed for expression of the proteins.
Preferably, the gp120 is expressed in mammalian cells that provide the same glycosylation and disulfide bonds as in native gp120. Expression of gp120 and fragments of gp120 in mammalian cells as fusion proteins incorporating N-terminal sequences of Herpes Simplex Virus Type 1 (HSV-1) glycoprotein D (gD-1) is described in Lasky, L. A. et al., 1986 (Neutralization of the AIDS retrovirus by antibodies to a recombinant envelope glycoprotein) Science 233: 209-212 and Haffar, O. K. et al., 1991 (The cytoplasmic tail of HIV-1 gp160 contains regions that associate with cellular membranes.) Virol. 180:439-441, respectively. A preferred method for expressing gp120 is described in the examples. In the examples, a heterologous signal sequence was used for convenient expression of the protein. However, the protein can also be expressed using the native signal sequence.
An isolated, purified gp120 polypeptide having one of the amino acid sequences illustrated in Table 1 can be produced by conventional methods. For example, the proteins can be chemically synthesized. In a preferred embodiment, the proteins are expressed in mammalian cells using an expression construct of this invention. The expressed proteins can be purified by conventional means. A preferred purification procedure is described in the examples.
gp120 Fragments
The present invention also provides gp120 fragments that are suitable for use in inducing antibodies for use in a vaccine formulation. A truncated gp120 sequence, as used herein, is a fragment of gp120 that is free from a portion of the intact gp120 sequence beginning at either the amino or carboxy terminus of gp120. A truncated gp120 sequence of this invention is free from the C5 domain. The C5 domain of gp120 is a major immunogenic site of the molecule. However, antibodies to the region do not neutralize virus. Therefore, elimination of this portion of gp120 from immunogens used to induce antibodies for serotyping is advantageous.
In another embodiment, the truncated gp120 sequence is additionally free from the carboxy terminal region through about amino acid residue 453 of the gp120 V5 domain. The portion of the V5 domain remaining in the sequence provides a convenient restriction site for preparation of expression constructs. However, a truncated gp120 sequence that is free from the entire gp120 V5 domain is also suitable for use in inducing antibodies.
In addition, portions of the amino terminus of gp120 can also be eliminated from the truncated gp120 sequence. In particular, the truncated gp120 sequence can be free from the gp120 signal sequence. The truncated gp120 sequence can be free from the carboxy terminus through amino acid residue 111 of the gp120 C1 domain, eliminating most of the C1 domain but preserving a convenient restriction site. However, the portion of the C1 domain through the V2 cysteine residue that forms a disulfide bond can additionally be removed, so that the truncated gp120 sequence is free from the carboxy terminus through amino acid residue 117 of the gp120 C1 domain. In a preferred embodiment, the truncated gp120 sequence is free from the amino terminus of gp120 through residue 111 of the C1 domain and residue 453 through the carboxy terminus of gp120.
The truncated gp120 sequences can be produced by recombinant engineering, as described previously. Conveniently, DNA encoding the truncated gp120 sequence is joined to a heterologous DNA sequence encoding a signal sequence.
It is understood that the application of the teachings of the present invention to a specific problem or situation is within the capabilities of one having ordinary skill in the art in light of the teachings contained herein. Examples of the products of the present invention and representative processes for their isolation, use, and manufacture appear below, but should not be construed to limit the invention. All literature citations herein are expressly incorporated by reference.
EXAMPLES Materials and Methods
Specimen collection from human volunteers. Blood was collected from MN-rgp120-immunized individuals who were infected with HIV-1 while participating in Phase I (NIH Protocol AVEG 016) and Phase II (NIH Protocol AVEG 201) HIV-1 vaccine trials sponsored by the National Institutes of Health (NIH). The demographics of the subjects in the study, and the study design have been described in McElrath; Seminars in Cancer Biol. 6:1-11 (1995); McElrath et al.; Abstracts from Eighth Annual Meeting of the National Cooperative Vaccine Development Groups for AIDS. Bethseda, Md. 216 (1996). Specimens were obtained according to an informed consent protocol approved by the institutional review boards of the participating institutions. In the experimental section, the time of HIV-1 infection is specified with regard to data provided by the NIH AIDS Vaccine Evaluation Network where PCR (RNA) and/or serologic assays were used to detect HIV-1 infection.
Sample preparation for cloning HIV-1 envelope glycoproteins. Peripheral blood mononuclear cells (PBMCs) from HIV-1 infected vaccinees were prepared from heparinized venous blood by gradient centrifugation using FICOLL-HYPAQUE leukocyte seperation medium. Cell number and viability were determined. After separation, PBMCs were washed twice in phosphate-buffered saline and suspended at a cell density of 6×106 cells/ml in PCR lysis buffer (50 mM KCl, 10 mM Tris (pH 8.4), 2.5 mM MgCl2, 0.1 mg/ml gelatin (Sigma), 0.45% NONIDET P40 detergent, 0.45% TWEEN 20 detergent (both detergents are commercially available from United States Biochemical Corp.) and 0.06 mg/ml Proteinase K (Gibco BRL) to lyse the cells. The lysate was incubated at 50-60° C. for 1 hour, followed by inactivation of the Proteinase K at 95° C. for 10 minutes. Lysates were shipped frozen and stored at -70° C. until use.
Polymerase chain reaction (PCR) amplification. Samples were subjected to two rounds of PCR amplification using the nested primers described below. In the first round, 25 μl aliquots of PBMC lysates (containing about 1 μg genomic DNA) were mixed with an equal volume of a PCR reaction mix containing 400 μM each dNTP, 200 μg/ml BSA (Sigma Chemical Corporation, RIA grade) and about 100 pmoles of each primer in 50 mM KCl, 20 mM Tris (pH 8.4) and 3 mM MgCl2. After an initial 10 minute denaturation step at 95° C., 5 units of Taq polymerase (AMPLITAQ, Perkin Elmer Cetus) were added during an 55° C. soak step, and samples were overlayed with mineral oil.
The PCR profile was as follows: 2 cycles having 1 minute at 55° C., 2.5 minutes at 72° C. and 1 minute at 94° C., followed by 28 cycles with 30 seconds at 55° C., 2.5 minutes at 72° C. and 45 seconds at 94° C., and an extension step at 72° C. for 5 minutes.
Aliquots of 10 μl from the first-round reactions were re-amplified with appropriate nested primers in a final reaction volume of 100 μl, using either the reagents and profile described above or the reagents and profile described in the PCR Optimizer Kit (Invitrogen.) PCR reaction products were purified using QIAQUICK-spin columns (Qiagen Inc.) The primer pair used in the first round was either 120.os.F (5'-gggaattcggatccAGAGCAGAAGACAGTGGCAATGA with homologous sequence at position 6248-6270 of HIVPV22) (SEQ. ID. NO:47) or JM11A (5'-ctcgag-CTCCTGAAGACAGTCAGACTCATCAAG at position 6048-6074) (SEQ. ID. NO:48) in the forward direction [Kusumi et al.; J. Virol. 66:875 (1992)] combined with 120.os.R (5'-ggtctagaagctttaGCCCATAGTGCTTCCTGCTGCT-CC at position 7836-7859) (SEQ. ID. NO:49) in the reverse direction. The internal nested primers were 120.BX.F (5'-gggcggatcctcgaGGTACCTGTRTGGAAAGAAGCA at position 6389-6410; R: A or G) (SEQ. ID. NO:50) and 120.is.R (5'-ggtctagaagctttaTGCTCCYAAGAACCCAAGGAACA at position 7819-7841; Y: T or C) (SEQ. ID. NO:51). Heterologous primer sequences are shown in lower case letters.
Subcloning of PCR products and the expression of recombinant envelope glycoproteins as fusion proteins. The HIV-1 envelope glycoprotein gp120 sequences were cloned and expressed as chimeric genes and fusion proteins, where the signal sequence and 27 amino acids from the mature N terminus of herpes simplex virus type 1 (HSV-1) were fused to the N-terminal sequences of the gp120 genes, corresponding to amino acid 13 of the mature gp120 sequence. PCR products containing gp120 sequences from the breakthrough specimens were cloned into pRK5 expression plasmid as chimeric genes using combinations of restrictions sites engineered into the heterologous PCR primer tails and the Xho I site engineered into the N-terminal sequence of HSV-1 gD.
The resulting double-stranded DNA was sequenced with Sequenase and the dGTP Reagent Kit (United States Biochemical Corp.). Sequences from glycoprotein D were provided to enhance expression and to provide a flag epitope to facilitate protein analysis, as described in Berman et al.; J. Virol. 7:4464-9 (1992); Nakamura et al.; AIDS and Human Retroviruses 8:1875-85 (1992); and Nakamura et al.; J. Virol. 67:6179-91 (1993).
Briefly, isolated DNA fragments generated by the PCR reaction were ligated into a plasmid (pRK.gD-5, pRKgDstop) designed to fuse the gp120 fragments, in frame, to the 5' sequences of the glycoprotein D (gD) gene of Type 1 Herpes Simplex Virus (gD-1)and the 3' end to translational stop codons. The fragment of the gD-1 gene encoded the signal sequence and 25 amino acids of the mature form of HSV-1 protein. To allow for expression in mammalian cells, chimeric genes fragments were cloned into the pRK5 expression plasmid (Eaton et al., Biochemistry 291:8343-8347 (1986)) that contained a polylinker with cloning sites and translational stop codons located between a cytomegalovirus promotor and a simian virus 40 virus polyadenylation site.
The resulting plasmids were transfected into the 293s embryonic human kidney cell line (Graham et al., J. Gen. Virol. 36:59-77 (1977)) using a calcium phosphate technique (Graham et al., Virology 52:456-467 (1973)). Growth conditioned cell culture media was collected 48 hr after transfection, and the soluble proteins were detected by ELISA or by specific radioimmunoprecipitation where metabolically labeled proteins from cell culture supernatants were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (PAGE) and visualized by autoradiography as described in Berman et al., J. Virol. 63:3489-3498 (1989) and Laemmli, Nature 227:680-685 (1970).
Serologic assays. Sera were assayed for antibodies to rgp120, antibodies to synthetic gp120 V3 domain peptides corresponding to sequences from the gp120 V3 domain, and antibodies able to inhibit the binding of MN-rgp120 to cell surface CD4 using serologic assays described in Berman et al.; J. Virol. 7:4464-9 (1992); Nakamura et al.; AIDS and Human Retroviruses 8:1875-85 (1992); and Nakamura et al.; J. Virol. 67:6179-91 (1993). Endpoint titers of antibody binding to gp120 and V3 peptides were determined using three fold-serial dilutions of sera. The endpoint dilution titer was defined as the last dilution that produced an optical density value that was two times higher than the mean of the optical densities of 1:50 diluted, pooled, normal human sera. Antibody titers were calculated by a computer program that interpolated values between antibody dilutions. The inter-assay coefficient of variation of positive control standard sera was 35%.
Binding of monoclonal antibodies to rgp120 from breakthrough viruses. An ELISA similar to that described by Moore et al.; AIDS 3:155-63 (1989) was used to measure the binding of various monoclonal antibodies (MAbs) to rgp120s from breakthrough viruses. Briefly, Nunc-Immuno plates (Maxisorp, certified) were coated (100 μl at 5 μg/ml in PBS at 4° C. overnight) with an affinity-purified sheep polyclonal antiserum to a peptide at the C terminus of gp120 (D7324, International Enzymes, Fallbrook, Calif.). After washing once with PBS-0.05% TWEEN-20 detergent, the plates were blocked with PBS-1.0% BSA for 30-60 minutes at room temperature. Cell culture supernatants from 293s cells, diluted to contain equivalent amounts of the gD-rgp120 fusion protein, were added and incubated for 2 hours at room temperature followed by three washes with PBS-0.05% TWEEN-20 detergent. Various MAbs were diluted in PBS-1.0% BSA and 100 μL of the diluted MAbs were added to each well and incubated for 1 hour at room temperature.
The plates were washed 3 times and incubated with 100 μl of a horseradish peroxidase-conjugated second antibody (goat anti-mouse or anti-human IgG, Cappel) for 1 hour at room temperature. After 3 washes the plates were developed and the OD492 (optical density at 492 nm) read in a plate reader. Growth conditioned cell culture supernatants were normalized by dilution based on binding by MAb 5B6 which is specific for HSV-1 glycoprotein D fusion protein.
Virus neutralization assays. The ability of vaccinee sera to inhibit infection of MT4 cells by HIV-1MN was measured in a cytopathicity assay where cell viability was quantitated using a calorimetric indicator dye, as described in Robertson et al.; J. Virol. Methods 20:195-202 (1988). Briefly, a virus stock of HIV-1MN (obtained from Dr. Michael Norcross, U.S. Food and Drug Administration) was prepared as the clarified supernatant from chronically infected H9/HIV-1MN cell culture. H9 cells chronically infected with HIV-MN were pelleted and resuspended in one-tenth the original volume of medium. Cell-associated virus was released by the mechanical shearing effects of rapid vortexing of the cells as described in Wrin et al.; J. Virol. 69:39-48 (1995).
An amount of virus sufficient to ensure complete cell lysis killing in 7 days was incubated with three-fold serial dilutions of test antisera, and then used to challenge MT4 T-lymphoid cells in 10% FCS/RPMI-1640 cell culture media. The cultures were incubated for 7 days at 37° C. in 5% CO2, and then cell viability was tested by the dye MTT, as described by Robertson et al.; J. Virol. Methods 20:195-202 (1988). Virus neutralization endpoints were quantitated by measurement of OD at 570-650 nm, and then the endpoint titers were calculated as the reciprocal of the antiserum dilution giving a signal that was two-fold above the control signal with unprotected (killed) cells. These titers were typically twice those calculated at 50% protection.
RESULTS
Immunization history of infected subjects. Since 1992, 499 adults have been immunized with MN-rgp120 in Phase I trials in low or moderate risk individuals and in a Phase II clinical trial involving moderate to high risk individuals. The studies described herein entail the genetic and immunologic characterization of the first seven of nine individuals who became infected with HIV-1 through high risk behavior during the course of these trials. A listing of the trials and summary of the status of the vaccinees is presented in Table 2A. A listing of the analysis of the vaccinees is presented in Table 2B.
              TABLE 2A                                                    
______________________________________                                    
Description of Vaccinees Infected with HIV-1                              
After Immunization with MN-rgp120                                         
                               ‡Antigen dose/                  
Study No.                                                                 
         Case No.    *Risk Group                                          
                               Adjuvant                                   
______________________________________                                    
016      C6          M/H       300/QS21                                   
016      C8          M/H       600/QS21                                   
016      C15         M/H       300/QS21                                   
201      C7          M/H       600/Alum                                   
201      C11         M/H       600/Alum                                   
201      C10         M/IDU     600/Alum                                   
201      C17         M/IDU     600/Alum                                   
______________________________________                                    
 *M/H indicates male homosexual; M/IDU indicate male intravenous drug user
 ‡numbers indicate dose in micrograms of MNrgp120 injected per 
 immunization; QS21 indicates antigen was formulated in QS21 adjuvant; Alu
 indicates MNrgp120 formulated in aluminum hydroxide.                     

              TABLE 2B                                                    
______________________________________                                    
Description of Vaccinees Infected with HIV-1                              
After Immunization with MN-rgp120                                         
        Injection                                                         
                 Injections Time of                                       
                                   ¤Interval:                    
Case    Schedule before     HIV-1+ to HIV-1+                              
No.     (months) HIV-1+     (months)                                      
                                   (months)                               
______________________________________                                    
 C6       0,1,10.5 2          4.00   2.00                                   
 C8       0,1      2          4.00   3.00                                   
  C15       0,1,2    3          6.25   4.00                                   
 C7       0,1,6,12 3          9.25   3.00                                   
 C11      0,1,6,12 4          19.50  6.75                                   
 C10      0,1,6,19 3          19.50  13.50                                  
 C17      0,1,6,18 4          24.75  6.25                                   
______________________________________                                    
 ¤indicates interval between last immunization and detection of  
 HIV1 infection.
Three of the infections occurred in a Phase I trial (NIH Protocol AVEG 201) that compared the safety and immunogenicity of MN-rgp120 formulated in two different adjuvants (alum and QS21), and four of the infections occurred in a Phase II trial aimed at establishing the safety and immunogenicity of MN-rgp120 in various high risk groups (e.g., intravenous drug users, homosexual and bisexual males, and partners of HIV-1 infected individuals).
Of the seven infections studied (Table 3), two (C6 and C8) occurred after two injections, three (C7, C10 and C15) occurred after three injections, and two (C11 and C17) occurred after receiving the four scheduled injections. The interval between receiving the last immunization and becoming infected was 2 to 13.5 months.
              TABLE 3                                                     
______________________________________                                    
Peak Post Boost MN-rgp120 Antibody Titers                                 
in Vaccinees that Became Infected with HIV-1                              
Injections                                                                
        C6      C8      C15  C7   C11   C10   C17                         
______________________________________                                    
1       <50     2185    79   <50  1890  na    na                          
2       21539   10125   na   413  32696 7771  7056                        
3       #       #       4460 9707 34728 11627 1841                        
                                              3                           
4       #       #       #    #    #     #     1134                        
                                              0                           
______________________________________                                    
 # indicates specimen not analyzed because of HIV1 infection.             
 na  indicates the sample was not available for testing.                  
 boldface  indicates unusually low antibody titers.
Antibody response to gp120 in vaccinated individuals. The magnitude and specificity of the antibody response to MN-rgp120 was measured by ELISA in all infected individuals throughout the course of the immunization regime (FIG. 1). Five of the seven subjects exhibited normal antibody response kinetics that included a small but reproducible primary response (1:100-1:2,000) and a strong secondary (booster) response (titters ranging from 1:7,000-1:32,000), and antibody responses following third and fourth injections that were similar or marginally higher than those achieved after the second immunization (FIG. 1, Table 3).
The antibody response observed in C7 (FIG. 1C) was unusual in that no antibodies were detectable after the primary injection and a titer of only 1:350 was detected after the second injection. It thus appeared that C7 did not respond to the primary immunization, and that the antibody response obtained after the second injection represented a primary immune response. Consistent with this hypothesis, the third injection elicited a titer of only 1:9,707, typical of those normally seen after two immunizations.
An atypical antibody response was also seen in subject C15 (FIG. 1G) who was immunized according to an accelerated immunization schedule of 0, 1, and 2 months. As expected, the antibody titer seen in this subject (1:4,460) was at the low end of what is typically achieved after two immunizations and was far below normal values for three immunizations. The lack of an effective booster response after the third immunization of C15 was not surprising in view of previous studies where an accelerated 0, 1, and 2 month immunization schedule in baboons [Anderson et al.; J. Infect. Dis. 160:960-9 ((1989)] similarly prolonged the secondary response and failed to elicit an effective tertiary booster response.
Retrospective analysis of serum and plasma from subjects C6 (FIG. 1A) and C8 (FIG. 1B) indicated that they became infected with HIV-1 at some point between the second and third immunizations. Serologic evidence of HIV-1 infection was evident in the gp120 antibody assays where the titers failed to decline two weeks after the second injection and instead formed an uncharacteristic high titer plateau (FIGS. 1A and 1B). A similar plateau in MN-rgp120 titer after the third injection, suggested that subject C7 became infected around week 36, approximately 16 weeks after receiving the third injection (FIG. 1C). Subjects C10 (FIG. 1E), C11 (FIG. 1D), C15 (FIG. 1G), and C17 (FIG. 1F) developed unexpected increases in gp120 titers, typical of HIV-1 infection, after either the third or fourth immunizations. The data obtained demonstrate that immunologic priming for MN-rgp120 antibody responses is insufficient to provide universal protection from HIV-1 infection.
Antibody titers to the V3 domain. To further characterize the antibody response to gp120, antibody titers were measured to a synthetic V3 domain peptide of MN-rgp120 containing the principal neutralizing determinant (PND). Five of the seven subjects developed good V3 titers (1:400 to 1:4000) after the second immunization, however two subjects (C7 and C15) required three immunizations before developing significant tiers (FIGS. 1C and 1G). As had been observed previously (11), the peak V3 titers in some individuals (e.g. C11, C10, C17) appeared to decline with each successive immunization (FIGS. 1D, 1E, and 1F). After HIV-1 infection, two patterns of V3 reactivity were observed. Three subjects (C6, C7, and C10) showed large increases in titer to V3 domain peptides (FIGS. 1A, 1C, and 1E) whereas C8 (FIG. 1B) showed a large decrease in V3 titer. At the time of analysis, the data were insufficient to draw any conclusions regarding the changes in V3 titers in response to HIV-1 infection in subjects C11, C15 and C17.
The results obtained indicate that the ability to form antibodies reactive with the V3 domain at various time-points prior to HIV-1 infection is not a valid correlate of protective immunity against all strains of HIV-1.
CD4 Inhibition titers. Antibodies that block the binding of gp120 to CD4 represent a heterogeneous class of virus neutralizing antibodies. Some are known to bind to the C4 domain of gp120 [Nakamura et al.; J. Virol. 67:6179-91 (1993); Anderson et al.; J. Infect. Dis. 160:960-9 ((1989)], and some are known to recognize conformation dependent discontinuous epitopes [Berman et al.; J. Virol. 7:4464-9 (1992); Nakamura et al.; J. Virol. 67:6179-91 (1993); McKeating et al.; AIDS Research and Human Retroviruses 8:451-9 (1992); Ho et al.; J. Virol. 65:489-93 (1991); Barbas et al.; Proc. Natl. Acad. Sci. USA 91:3809-13 (1994)].
One way to detect antibodies to both types of epitopes is to measure the ability of vaccinee sera to prevent the binding of [125 I]-labeled gp120 to cell surface CD4 [[Nakamura et al.; AIDS and Human Retroviruses 81875-85 (1992); Nakamura et al.; J. Virol. 67:6179-91 (1993)]. CD4 blocking titers were detected in all seven of the vaccinees prior to infection (FIG. 2) with peak titers that ranged from 1:10-1:300. At the last time point prior to infection, the CD4 titers in five of the seven vaccinees was low (1:30 or less). One vaccinee (C17), however, possessed a CD4 blocking titer of about 1:300 prior to infection (FIG. 2F). Thus, the lack of antibodies that block the binding of MN-rgp120 to CD4 cannot account for all of the infections. Large increases in CD4 blocking titers (1:100-1:1,000) were seen in five of the seven subjects after HIV-1 infection. These included vaccinees C6, C7, C8, C10, and C11. These results demonstrate that the CD4 blocking titers elicited by MN-rgp120 were lower than those elicited by natural infection.
Virus neutralizing activity. The virus neutralizing activity of antisera from MN-rgp120-immunized subjects was measured using a calorimetric assay that measured the viability of MT-4 cells after incubation with antibody treated virus (HIV-1MN). Since the actual date of infection was not known for any of the breakthrough infections, and serum samples were collected infrequently, the magnitude of the neutralizing antibody response at the time of infection is not known for any of the vaccinees.
Of the seven infections examined, the serum sample closest to the time of infection was that obtained from C7, where a neutralizing titer of 1:15 to HIV-1 was present three weeks prior to detection of HIV-1 infection (Table 4). In all other cases, however, the interval between the last injection and the time of infection was 10 to 25 weeks.
              TABLE 4                                                     
______________________________________                                    
Neutralization Activity of Sera from Vaccinees                            
Infected with HIV-1                                                       
Week  C6     C8      C15   C7    C11   C10   C17                          
______________________________________                                    
0     <10*   <10*    <10*  <10*  <10*  <10*  <10*                         
2     <10    <10     <10   --    --    --    --                           
4     <10*   <10*    nd*   <10*  <10*  <10*  <10*                         
6     10     80      --    <10   30    150   150                          
8     --     --      nd*   --    --    --    --                           
10    --     --      35    --    --    --    --                           
15    --     --      --    <10   --    --    --                           
16    150#   250#    --    --    30    10    <10                          
24                   150#  <10*  20*   <10*  <10*                         
26                         70    500   200   400                          
30                         --    --    40    100                          
33                         15    --    --    --                           
35                         --    100   --    --                           
36                         30#   --    10    40                           
52                               30*   <10   <10                          
54                               250   --    --                           
57                               100   --    --                           
63                               90    --    --                           
64                               --    --    <10                          
77                               40#   --    --                           
78                                     500#  10*                          
80                                           100                          
84                                           60                           
90                                           150                          
104                                          150#                         
______________________________________                                    
 *  indicates immunization.                                               
 # indicates HIV1 positive.                                               
 nd  indicates not done.                                                  
 -- indicates sample not available.
When sera from the two early infections were examined (Table 4), one individual (C6) had a peak neutralizing titer of 1:10 ten weeks prior to detection of HIV-1 infection, whereas the other individual (C8) had a neutralizing titer of 1:80 ten weeks prior to detection of HIV-1 infection. Subject C15, who was immunized according to an accelerated immunization schedule, developed a neutralizing titer of 1:35 after the third injection, 14 weeks prior to HIV-1 infection. Subject C10, who had a peak neutralizing titer of 1:200 following the third immunization (week 24), had no detectable titer at week 52, six months prior to the first indication of HIV-1 infection (week 78).
Subject C11 possessed a neutralizing titer of 1:90 at fourteen weeks prior to detection of HIV-1 and a peak titer of 1:500 following the third immunization. Similarly vaccinee C17 had a neutralizing titer of 1:150 fourteen weeks prior to infection and a peak titer of 1:400 at two weeks after the third immunization.
Based on the rate of decay of the gp120 response of approximately two months [Belshe et al.; JAMA 272(6):475-80 (1994)], as well as the observation that neutralizing titers of 1:150 decayed to 1:10 in 10 weeks in vaccinees C10 and C17, it appears that neutralizing titers in C8, C15, C11, and C17 could have declined to 1:10 or less in the intervals between the last pre-infection serum sample and the time of HIV-1 detection.
The results of these studies demonstrated that all vaccinees developed some level of virus-neutralizing antibodies at some time prior to HIV-1 infection, and that the magnitude of the neutralizing response was probably low at the time of infection. In general, the magnitude of the virus-neutralizing response observed in the individuals that became infected with HIV-1 was comparable to that seen in non-infected vaccinees as described in Belshe et al.; JAMA 272(6):475-80 (1994).
Sequences of Viruses. To evaluate the similarity of the breakthrough viruses with the vaccine antigen, nucleotide sequences for gp120 from all seven breakthrough viruses were determined. Envelope glycoprotein genes were amplified from proviral DNA using the polymerase chain reaction. Sequences were obtained by direct amplification of DNA from lysates of gradient-purified lymphocytes obtained directly from patient blood without any intermediate tissue culture or amplification step.
A listing of the complete gp120 sequences (two clones per specimen) is provided in FIG. 3. All seven envelope glycoproteins possessed sequences typical of subtype (clade) B viruses. The overall homology with MN-rgp120 ranged from 69-80% (Table 5).
                                  TABLE 5                                 
__________________________________________________________________________
Comparison of MN-rgp120 Sequence with Sequences                           
from Infected Vaccinees*                                                  
MN       C6.1                                                             
             C8.3                                                         
                 C7.2                                                     
                     C11.5                                                
                          C10.5                                           
                               C17.1                                      
                                    C15.2                                 
__________________________________________________________________________
MN   100 79  78  70  75   69   80   72                                    
C6.1     100 78  70  81   75   90   79                                    
C8.3         100 68  80   76   84   83                                    
C7.2             100 80   73   76   73                                    
C11.5                100  75   70   80                                    
C10.5                     100  70   72                                    
C17.1                          100  87                                    
C15.2                               100                                   
__________________________________________________________________________
 *  Data indicate percent identity.
Interestingly, a high percentage (four of seven) of the breakthrough viruses differed from MN-rgp120 by 25-30% [Myers et al.; Retroviruses and AIDS Database, Los Alamos National Laboratory (1992 and 1995)]. Historically this degree of sequence variation is typical of inter-subtype (intra-clade) variation rather than intra-subtype variation which is expected to be in the 10-20% range [Myers et al.; Retroviruses and AIDS Database, Los Alamos National Laboratory (1992 and 19950]. Of the viruses with the greatest homology to MN-rgp120, two (C6 and C8) occurred as early infections, prior to complete immunization, and one (C17) occurred as a late infection.
Polymorphism in the V3 Domain. Of particular interest were polymorphisms in regions known to contain epitopes recognized by virus neutralizing antibodies. The best characterized neutralizing epitope, the principal neutralizing determinant (PND), occurs at the tip of the V3 loop. In subtype B viruses, approximately 60% possess the MN serotype-defining signature sequence, IGPGRAF (SEQ. ID. NO:52), based on identity with the prototypic MN strain of HIV-1 [Berman et al.; J. Virol. 7:4464-9 (1992); Myers et al.; Retroviruses and AIDS Database, Los Alamos National Laboratory (1992 and 1995); La Rosa et al.; Science 249:932-5 (1990)].
Three of the viruses (C6, C8, and C17) possessed the MN serotype signature sequence (FIG. 3). In contrast, four viruses possessed sequences with radical amino acid substitutions in the PND [IGPGRAW (C7), LGPGSTF (C11), IGPGRVL (C10), and IGPGSAF (C15)] (SEQ. ID. NOs:53-56, respectively), and therefore were classified as "non-MN like" viruses. Of note, each of the four "non-MN-like" sequences were rare (Table 6) and were not typical of the most common "non-MN" variants of subtype B viruses [Myers et al.; Retroviruses and AIDS Database, Los Alamos National Laboratory (1992 and 1995)].
              TABLE 6                                                     
______________________________________                                    
Frequency of Polymorphisms at the Principal                               
Neutralizing Determinant in HIV-1 Infected                                
Individuals Immunized with MN-rgp120*                                     
        Ob-                                                               
        served                                                            
              Dataset Frequency                                           
V3 Sequence                                                               
          Fre-    GNE     LANL   LANL.1 LaRosa                            
Sequence                                                                  
        n     quency  (n = 52)                                            
                            (n = 519)                                     
                                   (n = 160)                              
                                          (n = 245)                       
______________________________________                                    
GPGRAF  3     0.42    0.67  0.57   0.66   0.60                            
GPGRAW  1     0.14    0.03  0.013  0.06   0.010                           
GPGRVL  1     0.14    <0.02 0.004  <0.006 <0.008                          
GPGSTF**                                                                  
        1     0.14    <0.02 <0.002 <0.006 <0.004                          
GPGSAF  1     0.14    0.02  0.011  <0.006 <0.004                          
______________________________________                                    
 *Data set GNE refers to a collection of 52 independent isolates collected
 in 1992; dataset LANL refers to a collection of 519 sequences reported by
 Myers et al., Retroviruses and AIDS Database, Los Alamos National        
 Laboratory 1992 and 1995; LANL.1 refers to a collection of 160           
 epidemiologically unlinked individuals provided by B. Korber (personal   
 communication); dataset La Rosa refers to sequence data reported by La   
 Rosa et al., Science 249:9325 (1990).                                    
 **Sequences were not present in the data sets examined.
The prevalence of viruses with PND sequences matching the breakthrough viruses ranged from a high of 1.3% (C7) to a low of 0.2% (C11) in a listing of 519 subtype B sequences compiled by the Los Alamos National Laboratory [Myers et al.; Retroviruses and AIDS Database, Los Alamos National Laboratory (1992 and 1995)]. Similarly low frequencies were observed in three other independently derived data sets (Table 6). The occurrence of these sequences did not differ significantly between data sets collected prior to 1985 [La Rosa et al.; Science 249:932-5 (1990)] and data collected 1992, or from a set of 160 epidemiologically unlinked individuals (B. Korber, personal communication). All four sets of data agreed that the prevalence of viruses with MN-like PND sequences was in the range of 60%. Based on this data, four of the seven breakthrough infections were determined to be caused by viruses that fell outside of the spectrum of viruses that the vaccine was expected to prevent.
Other features of breakthrough virus V3 domains. Like MN-rgp120, the V3 domains of all of the breakthrough viruses were 36 amino acids in length. However, all seven viruses differed from MN-rgp120 with respect to the number of glycosylation sites and with respect to the syncytium-inducing (SI) signature sequence.
The sequence of MN-rgp120 is somewhat unusual [Myers et al.; Retroviruses and AIDS Database, Los Alamos National Laboratory (1992 and 1995)] in that it lacks an N-linked glycosylation site at position 306 in the V3 domain. The lack of this glycosylation site does not appear to be antigenically significant since antisera to MN-rgp120 are known to neutralize a variety of viruses (e.g. SF-2, DU6587-5, DU4489-5, CC) that possess a glycosylation site at this position [Berman et al.; J. Virol. 7:4464-9 (1992)].
In addition, the V3 domain of MN-rgp120 possessed sequence polymorphisms (R at position 311, K at position 324, K at position 328) typical of syncytium inducing viruses [Fouchier et al.; J. Virol. 66:3183-87 (1992)], whereas all seven breakthrough viruses possessed sequences associated with non-syncytium-inducing viruses. Syncytium-inducing viruses have been associated with rapid disease progression [Tersmette et al.; J. Virol. 62:2026-32 (1988)] and T cell tropism [O'Brien et al.; Nature (London) 348:69-73 (1990); Shioda et al.; Nature (London) 349:167-9 (1991)]. To date viruses with these properties have not been recovered from any of the MN-rgp120 immunized volunteers.
Polymorphism in the V1, V2 and C4 domains. Previous investigations have identified additional neutralizing epitopes in the V1, V2 and C4 domains of gp120 [Nakamura et al.; J. Virol. 67:6179-91 (1993); McKeating et al.; AIDS Research and Human Retroviruses 8:451-9 (1992); Ho et al.; J. Virol. 65:489-93 (1991); Barbas et al.; Proc. Natl. Acad. Sci. USA 91:3809-13 (1994); McKeating et al.; J. Virol. 67:4932-44 (1993); Moore et al.; J. Virol. 67:6136-6151 (1993); Davis et al.; J. Gen. Virol. 74:2609-17 (1993)].
The best characterized of these neutralizing epitopes is in the C4 domain which has attracted special attention because antibodies binding to this area are known to block the binding of gp120 to CD4 [Moore et al.; AIDS 3:155-63 (1989); McKeating et al.; AIDS Research and Human Retroviruses 8:451-9 (1992)]. Because the epitope is located in a conserved (C) domain, naturally-occurring polymorphism in this region is far more limited than in other neutralizing epitopes. Nakamura et al.; J. Virol. 67:6179-91 (1993) reported that the binding of a number of neutralizing MAbs was dependent on K at position 429.
Comparison of the sequence of MN-rgp120 with other strains of HIV-1 showed that a common polymorphism, involving the substitution of E for K, occurs at this position. Indeed, substrains of the same virus isolate often show polymorphism at this position. The HXB2 substrain of HIV-1LAI contains K at position 429, whereas the BH10, IIIB, and LAV substrains of the HIV-1LAI contain E at this position [Nakamura et al.; J. Virol. 67:6179-91 (1993)]. Similarly, the 1984 isolate of HIV-1MN exhibited E at this position, while the 1990 isolate of HIV-1MN, used to produce MN-rgp120, possessed K at this position.
When the sequences of the infected vaccine recipients were examined (FIG. 3), the virus from subject C17, like MN-rgp120 contained K at position 429, whereas the six other viruses that differed from the vaccine immunogen possessed E at this position. These results demonstrated that six of the seven breakthrough viruses differed from the vaccine immunogen at the CD4-blocking, neutralizing epitope in the C4 domain of gp120.
Studies with monoclonal antibodies have defined neutralizing epitopes in the V1 and V2 domains of gp120 [McKeating et al.; J. Virol. 67:4932-44 (1993); Moore et al.; J. Virol. 67:6136-6151 (1993); Davis et al.; J. Gen. Virol. 74:2609-17 (1993)]. Like the polymorphisms that occur in the C4 domain, the V2 domains exhibit several common polymorphisms that affect the binding of virus neutralizing antibodies. One such polymorphism occurs at position 171 which is critically important for the binding of murine MAb 1025, whereas residue 187 is important for the binding of MAb several MAbs represented by 1088.
When the V2 domain sequences were examined (FIG. 3), all of the infected-vaccinee viruses differed from MN-rgp120 in that R replaced G at position 171 and I or V replaced E at position 187. Antibodies recognizing these adjacent sites in the V2 domain of MN-rgp120 would not be expected to neutralize viruses with radical amino acid substitutions at these position. Thus, all seven breakthrough viruses differed from MN-rgp120 at a neutralizing epitope in the V2 domain of gp120.
Other neutralizing epitopes have been reported in the V1 domain of gp120 [O'Brien et al.; Nature (London) 348:69-73 (1990); McKeating et al.; J. Virol. 67:4932-44 (1993)]. Although the neutralizing epitopes in the V1 domain of MN-rgp120 have not been characterized, the polymorphism seen among the breakthrough viruses in this region was interesting. Particularly striking (FIG. 3) was that the length of this domain ranged from 20 amino acids (C17) to 45 amino acids (C6), and the number of N-linked glycosylation sites ranged from 2 to 6. In contrast, the V1 domain of MN-rgp120 is 31 amino acids in length and encodes three N-linked glycosylation sites.
Although examination of sequence databases suggest that variation in the V2 region is comparable to the V1 region, the V2 region of the breakthrough viruses showed less variation than expected. Specifically, the length of the V2 region ranged from 36 amino acids (C7) to 39 amino acids in length, with six of seven viruses containing three N-linked glycosylation sites in this domain. A high degree of polymorphism was found in the V4 region where sequences ranged from 26 (C10) to 33 (C15, C7) amino acids in length and contained either 4 or 5 N-linked glycosylation sites.
Antigenicity of envelope glycoproteins from breakthrough viruses. To determine the significance of sequence variation on glycoprotein antigenicity, recombinant gp120 was prepared from the viruses of all seven infected vaccinees (FIG. 4). In these studies a series of MAbs was assembled and their binding to MN-rgp120 was compared to that of rgp120 from the vaccinee isolates by ELISA (Table 7).
              TABLE 7                                                     
______________________________________                                    
Relative Reactivity* of MAb Binding to rgp120 from                        
Infected Subjects Compared with Binding to MN-rgp120                      
       V3        Discontinuous                                            
                             C8     V2                                    
gp120    1034   50.1     1.5E 1025   1024 1088                            
______________________________________                                    
MN       1.0    1.00     1.00 1.00   1.00 1.00                            
C6.1     0.37   0.37     0.17 0.00   0.00 0.00                            
C6.5     0.33   0.33     0.75 0.00   0.00 0.00                            
C8.3     0.11   0.37     0.38 0.00   0.00 0.00                            
C8.6     0.14   0.34     0.29 0.00   0.00 0.00                            
C7.2     0.47   0.60     0.71 0.00   0.00 0.00                            
C11.5    0.00   0.00     0.17 0.00   0.00 0.00                            
C11.7    0.00   0.00     0.17 0.00   0.00 0.00                            
C10.5    0.33   0.40     0.46 0.24   0.03 0.04                            
C10.7    0.42   0.48     0.50 0.29   0.07 0.09                            
C17.1    0.33   0.52     0.33 0.00   0.30 0.07                            
C17.3    0.37   0.56     0.33 0.00   0.38 0.06                            
C15.2    0.00   0.47     0.92 0.00   0.00 0.00                            
C15.3    0.00   0.37     0.63 0.00   0.00 0.00                            
______________________________________                                    
 *Relative reactivity values represent ratio of optical densities obtained
 with rgp120 from patient isolates divided by optical density obtained for
 MNrgp120 at a MAb concentration of 2 micrograms per milliliter.
In control experiments, the binding of MAb 5B6 (which is specific for the HSV gD-1 flag epitope fused to the N terminus of all of the rgp120 protein) was used to standardize the amount of gp120 from each isolate (FIG. 5A). These studies demonstrated that the assay was carried out under conditions where equivalent amount of rgp120s were captured onto wells of microtiter plates.
The antigenic structure of the V3 domain was examined using the 1034 MAb (isolated from mice immunized with MN-rgp120 as described in Nakamura et al.; J. Virol. 67:6179-91 (1993) and the 50.1 MAb (prepared from mice immunized with a synthetic V3 domain peptide as described in Rini et al.; Proc. Natl. Acad. Sci. USA 90:6325-9 (1993). Both MAbs are known to exhibit potent virus neutralizing activity. When binding to the recombinant proteins was examined, the MAb binding to MN-rgp120 was at least 10-fold greater than to any of the breakthrough virus envelope proteins (FIGS. 5B and C). Surprisingly, rgp120 from the three patient isolates (C8, C6, and C17) that possessed the MN serotype-defining sequence, IGPGRAF (SEQ. ID. NO:32), varied from one another in their MAb binding activity. Thus, the binding of MAb 1034 and MAb 50.1 to rgp120 from C17 was significantly greater than the binding to rgp120s from C6 and C8.
A distinction in the epitopes recognized by these MAbs was evident since C6-rgp120 and C8-rgp120 gave comparable binding with 50.1, whereas 1034 bound better to the C6-derived protein than the C8-derived protein. The poorest MAb reactivity was with rgp120s from C11 and C15. This result was consistent with sequence analysis demonstrating that these two viruses both possessed the radical substitution of S for R at position 18 in the V3 domain. Surprisingly, both of these MAbs exhibited better than expected binding to rgp120 from the C7 and C10 viruses. Like MN-rgp120, both proteins contained the penta-peptide, IGPGR sequence (SEQ. ID. NO:57) in the V3 loop, but differed from MN-rgp120 in that V and L replaced A and F at positions 319 and 320 in gp120 from C10, and W replaced F at position 320 in gp120 from C7. These results indicate that R at position 318 is essential for the binding of these two MAbs, and that the epitopes recognized by 1034 and 50.1 are not completely destroyed by the hydrophobic substitutions at positions 319 and 320.
As predicted from the sequence data, there was little if any binding to the breakthrough virus rgp120s using MAbs (1088 and 1025) directed to the V2 region of MN-rgp120. Also consistent with sequence data was the observation that MAb 1024 directed to the C4 domain of MN-rgp120 gave some reactivity with C17-rgp120 which, like MN-rgp120 contained K at position 429, but gave no reactivity with the other isolates that contained E at residue 429.
Together, these studies demonstrated that the antigenic structure of all seven breakthrough viruses differed from the vaccine immunogen at three well characterized neutralizing epitopes.
A totally different pattern of reactivity was observed with the human hybridoma, MAb 15e, prepared from an HIV-1 infected individual as described in Ho et al.; J. Virol. 65:489-93 (1991). With this MAb, the greatest binding was achieved with MN-rgp120 and rgp120 from C7, and the poorest reactivity was seen with the two clones of rgp120 from the C11. Moderate, but comparable reactivity was seen with rgp120s from the C10 and C17.
These results demonstrate that the 15e epitope is polymorphic, and that the epitope is conserved on MN-rgp120 and rgp120 from C7, but has been lost on rgp120s from C11. Interestingly, the two different clones of gp120 derived from C6 gave strikingly different patterns of antibody binding. Thus, rgp120 from clone C6.5 exhibited strong reactivity with this antibody, whereas rgp120 from clones C6.1 exhibited significantly weaker activity with this MAb. Comparison of sequence data (FIG. 3) showed that the two C6 clones differed at 6 amino acid positions. Based on comparative binding to the other viral proteins of known sequence, it appeared that the substitution of K for I at position 351 in the C3 domain of gp120 could account for the difference in binding activity. This result is also consistent with both clones of C11 similarly containing a positively-charged K at this position, and also being poorly reactive with this MAb. Alternatively, a T for I substitution at position 439 in the C4 domain could account for the difference in 15e binding between C6.1 and C6.5. Although the inability of the two C11 clones to bind 15e cannot be explained by polymorphism at this position in the C4 domain, they could be affected by the adjacent T for M substitution at position 434.
DISCUSSION
In these studies, the viruses and immune responses in seven of nine vaccinees who became infected with HIV-1 through high risk activity while participating in Phase 1 or Phase 2 trials of MN-rgp120, a candidate HIV-1 vaccine were analyzed. Such infections would be expected to occur for one of two reasons: 1) lack of sufficient immune response at the time of infection; or 2) infection with viruses that fall outside of the antigenic spectrum expected to be covered by the vaccine immunogen. The data indicate that both explanations may be involved with the infections observed (Table 8).
              TABLE 8                                                     
______________________________________                                    
Summary of Breakthrough Infections                                        
           MN-rgp120                                                      
                   Homologous to MN-rgp120                                
Case  Adequate   Homology  V3    C4     V2                                
No.   Immunization                                                        
                 (%)       PND   Epitope                                  
                                        Epitope                           
______________________________________                                    
C6    -          79        +     -      -                                 
C8    -          78        +     -      -                                 
C15   -          72        -     -      -                                 
C7    -          70        -     -      -                                 
C11   +          75        -     -      -                                 
C10   +          69        -     -      -                                 
C17   +          80        +     +      -                                 
______________________________________
Two of the infections occurred in individuals who failed to receive the minimum three doses of vaccine typically required for the induction of protective immunity with protein subunit vaccines (e.g. hepatitis B virus formulated in alum adjuvant as described in Francis et al.; Ann. Int. Med. 97:362-6 (1982). Two additional breakthrough infections occurred in vaccinees who had weak or undetectable primary (C7) and booster (C15) responses. Of the three individuals who became infected with HIV-1 after receiving three or more productive immunizations (C10, C11, and C17), at least two, and possibly all three, appear to have become infected more than six months after receiving their last immunization. Because antibody titers to MN-rgp120 typically decay with a half-time of 2 to 2.5 months [Belshe et al.; JAMA 272(6):475-80 (1994); Berman et al.; AIDS 8:591-601 (1994)], antibody titers would be expected to have decayed at least eight-fold and possibly as much as sixty four-fold at the time of infection. Thus, the lack of a sufficient immune response at the time of infection represents a potential explanation for at least six of the seven breakthrough infections.
Data from vaccine efficacy studies in gp160 immunized chimpanzees [McElrath et al.; Longitudinal Vaccine-Induced Immunity and Risk Behavior of Study Participants in AVEG Phase II Protocol 201. In: Abstracts from Eighth Annual Meeting of the National Cooperative Vaccine Development Groups for AIDS. Bethseda, Md. 1996:216] challenged with HIV-1, and gp120-immunized rhesus macaques challenged with a chimeric SIV/HIV-1 virus (SHIV) suggest that the magnitude of the neutralizing antibody response at the time of infection is a critical correlate of protective immunity. If maintaining neutralizing antibody titers proves to be a valid correlate of protective immunity in humans, then formulations (e.g. novel adjuvants) or immunization regimes (frequent boosting) designed to maximize the antibody responses may be required to achieve long lasting protection. Use of a booster every six months may be advantageous.
The other likely explanation for the late infections is the antigenic difference between the vaccine and the breakthrough virus envelope glycoproteins. This explanation is supported by the observation that four of the seven breakthrough viruses possessed envelope glycoproteins that differed from the MN-rgp120 by 25-30% at the amino acid level. Differences of this magnitude have historically [Myers et al.; Retroviruses and AIDS Database, Los Alamos National Laboratory (1992 and 1995)] been associated with inter-subtype variation and far exceeds the average 10-20% variation expected for viruses within the same subtype.
Although the biologic significance of sequence variation in many regions of the envelope glycoprotein is unclear, polymorphism at neutralizing epitopes is an important factor that affects vaccine efficacy. Previous studies [Salmon-Ceron et al.; AIDS Res. and Human Retroviruses 11:1479-86 (1995); Javaherian et al.; Science 250:1590-3 (1990)] have demonstrated that the breadth of neutralizing activity that could be elicited by HIV-1 envelope derived vaccines was critically dependent on the sequence of epitopes in the V3 domain (e.g.; the PND). Thus, candidate vaccines based on the LAI strain of HIV-1 (the prototypic "non-MN-like" subtype B virus), exhibited little or no cross neutralizing activity with subtype B viruses, whereas vaccines that contained the "MN-like-" PND sequence (IGPGRAF) (SEQ. ID. NO:52) exhibited broad cross neutralizing activity. That four of the seven breakthrough viruses possessed envelope glycoproteins with radical amino acid substitutions in the PND is consistent with the explanation that differences in antigenic structure explain some of these infections.
Over the last few years, it has become clear that polymorphism among "MN-like" viruses occurs at neutralizing epitopes outside of the PND. The best example occurs in the C4 domain where two antigenically distinct variants are distinguished by the presence of either K or E at position 429 [Moore et al.; AIDS 3:155-63 (1989)]. Because six of the seven breakthrough viruses differed from the vaccine strain in that they contained E rather than K at position 429, antibodies raised to the C4 domain of MN-rgp120 were unlikely to neutralize the viruses infecting in six of the seven vaccinees.
Other neutralizing epitopes are known to be present in the V1 and V2 domains of gp120. Although these regions are highly variable, due to insertions and deletions, neutralizing epitopes have been described by McKeating et al.; J. Virol. 67:4932-44 (1993); Moore et al.; J. Virol. 67:6136-6151 (1993); and Davis et al.; J. Gen. Virol. 74:2609-17 (1993). Several of these epitopes overlap an amino terminal sequence of the V2 domain containing the tri-peptide sequence RDK at positions corresponding to 142 to 144 of MN-rgp120 [McKeating et al.; J. Virol. 67:4932-44 (1993); Moore et al.; J. Virol. 67:6136-6151 (1993)]. Like the C4 epitope, variation in this sequence is known to occur between different substrains derived from the same parental isolate. Since all seven breakthrough viruses differed from MN-rgp120 in that they possessed the RDK sequence, rather than the GDK sequence present in the vaccine antigen, neutralizing antibodies to the V2 domain of MN-rgp120 would not have been expected neutralize any of the viruses recovered from the vaccinees immunized with MN-rgp120.
Although polymorphisms at neutralizing epitopes might account for the lack of protection in most of the infections, this does not appear to explain the infection of vaccinee C17, who was infected by a virus that matched MN-rgp120 in the V3 and C4 domains. If a difference in sequence was responsible for the lack of protection in this case, the critical difference might relate to the unusual sequence in the V1 domain of gp120 from this breakthrough virus. Several studies have shown that the V1 domain possesses epitopes recognized by virus neutralizing monoclonal antibodies [McKeating et al.; J. Virol. 67:4932-44 (1993); Davis et al.; J. Gen. Virol. 74:2609-17 (1993); Kayman et al.; J. Virol. 68:400-410 (1994)].
Although far less is known about the V1 epitopes relative to other neutralizing sites, the V1 epitopes appear to be conformation-dependent, and antisera from HIV-1 infected individuals recognize epitopes in the V1 and V2 domains [McKeating et al.; J. Virol. 67:4932-44 (1993); Kayman et al.; J. Virol. 68:400-410 (1994)]. The V1 sequence of the virus from C17 is noteworthy because it is smaller and contains fewer N-linked glycosylation sites than that of MN-rgp120 or any of the other breakthrough viruses. By the same token, the envelope glycoproteins from C11 and C6 are noteworthy because they are significantly larger and contain more glycosylation sites than MN-rgp120 or the other breakthrough viruses.
While differences in amino acid sequence can provide clues to differences in antigenic structure, the consequences of such polymorphism can only be proven through antibody binding studies. To correlate differences in sequence with differences in antigenic structure, gp120 from two clones each of all seven breakthrough viruses was expressed and the antigenicity of the clones with a panel of monoclonal antibodies was examined. As predicted from the sequence data, none of the breakthrough virus envelope glycoproteins reacted with neutralizing MAbs to the V2 domain of MN-rgp120. When MAbs to the C4 domain were examined, only the C17 envelope glycoprotein (that matched MN-rgp120 with respect to K429) showed significant, albeit lower, binding. Surprisingly, the three breakthrough envelope glycoproteins that contained the subtype B PND consensus sequence, IGPGRAF (SEQ. ID. NO:52), gave poor reactivity with all three PND directed MAbs, even though they possessed PND sequences closely related to the vaccine immunogen. Thus, all three of the vaccinee isolates appeared to possess changes outside of the recognition site that interfered with MAb binding.
It has been known for many years that resistance to neutralization in vitro can sometimes be attributed to mutations in remote sequences that alter the conformation of neutralizing epitopes and interfere with recognition by virus neutralizing antibodies [Nara et al.; J. Virol. 64:3779-91 (1990); Cordonnier et al.; Nature 340:571-4 (1989)]. Together, these results indicate that the antigenic structure of the envelope glycoproteins recovered from the breakthrough viruses differed significantly from that of the vaccine antigen.
A novel result was the localization of residues in the C3 domain that appeared to affect the binding of the virus neutralizing human MAb, 15e. This MAb is known to recognize a discontinuous epitope, block CD4 binding, and neutralize a variety of laboratory and primary isolates of HIV-1 [Ho et al.; J. Virol. 65:489-93 (1991); Thali et al.; J. Virol. 66:5635-5641 (1992); Moore et al.; AIDS Res. Hum. Retroviruses 9:1179-1187 (1993)].
Comparative binding to envelope glycoproteins from the breakthrough viruses indicated that recognition by this antibody is critically dependent on residues in the C3 or C4 domains of gp120. The unique occurrence of a positively charged K at position 351 in the C3 domain provides a common explanation for the inability of the C11.5, C11.7 and C6.1 strains of HIV-1 to bind to 15e. Alternatively, it is possible that different amino acid substitutions in different locations account for the failure of 15e to bind to rgp120s from the C6 and C11 clones. The only obvious positions where substitutions of this type occur are in the C4 domain where T replaces M at 434 (C11) and T replaces I at 439.
The present studies demonstrate that the current formulation of MN-rgp120 is less than 100% effective against HIV-1 infection. Based on previous in vitro and in vivo studies with MN-rgp120, protection from natural HIV-1 infection in humans is expected to depend on a threshold concentration of virus-neutralizing antibodies, and antigenic similarity between the vaccine immunogen and the challenge virus.
In this regard, only one of the seven breakthrough infections (C17) was unexpected. This individual received a full course of immunizations yet became infected with a virus similar to MN-rgp120 at at least two important neutralizing epitopes (V3 and C4 domains). This infection might be related to the magnitude of the antibody response at the time of infection, or antigenic differences between the breakthrough virus and the vaccine strain, or circumstances of infection (e.g., ulcerative lesions, infection by donor with acute infection or high viremia), not monitored in this protocol. Alternatively this individual may represent a true vaccine failure, without clear explanation.
On balance, the analysis of breakthrough infections described herein did not uncover any data that would discourage the continued development of MN-rgp120 as a vaccine to prevent HIV-1 infection. The results support speculation that enhancing vaccine immunogenicity (as by additional booster immunizations) may be required to maintain long term protective immunity, and that the addition of rgp120 from other antigenically different strains of virus in addition to MN-rgp120 are useful to expand the breadth of protection.
The availability of viruses and viral glycoproteins derived from breakthrough infections may provide an important means to streamline the process of identifying new antigens for inclusion into a multivalent vaccine. Recombinant viral glycoproteins prepared from breakthrough viruses, by definition, possess antigenic structures that are significantly different from MN-rgp120, and are be representative of viruses currently being transmitted. Thus, combining rgp120 from breakthrough viruses with MN-rgp120 is an effective way complement and significantly expand antigenic complexity and increase breadth of cross neutralizing activity.
__________________________________________________________________________
#             SEQUENCE LISTING                                            
- <160> NUMBER OF SEQ ID NOS: 57                                          
- <210> SEQ ID NO 1                                                       
<211> LENGTH: 1503                                                        
<212> TYPE: DNA                                                           
<213> ORGANISM: HIV                                                       
<220> FEATURE:                                                            
<221> NAME/KEY: CDS                                                       
<222> LOCATION: (1)...(1503)                                              
- <400> SEQUENCE: 1                                                       
- ggg gta cct gtg tgg aag gaa gca acc acc ac - #t cta ttt tgt gca tca     
  48                                                                      
Gly Val Pro Val Trp Lys Glu Ala Thr Thr Th - #r Leu Phe Cys Ala Ser       
#                 15                                                      
- gat gct aaa gca tat gac aca gag gtg cat aa - #t gtt tgg gcc aca cat     
  96                                                                      
Asp Ala Lys Ala Tyr Asp Thr Glu Val His As - #n Val Trp Ala Thr His       
#             30                                                          
- gct tgt gta ccc aca gac cca aac cca caa ga - #a atg gta ttg gaa aat     
 144                                                                      
Ala Cys Val Pro Thr Asp Pro Asn Pro Gln Gl - #u Met Val Leu Glu Asn       
#         45                                                              
- gtg aca gaa gat ttt aac atg tgg aaa aat ga - #c atg gta gaa cag atg     
 192                                                                      
Val Thr Glu Asp Phe Asn Met Trp Lys Asn As - #p Met Val Glu Gln Met       
#     60                                                                  
- cat gag gat ata atc agt tta tgg gat caa ag - #c cta aaa cca tgt gta     
 240                                                                      
His Glu Asp Ile Ile Ser Leu Trp Asp Gln Se - #r Leu Lys Pro Cys Val       
# 80                                                                      
- aaa tta acc cca ctc tgt att act tta aat tg - #c acc aat tgg aag aag     
 288                                                                      
Lys Leu Thr Pro Leu Cys Ile Thr Leu Asn Cy - #s Thr Asn Trp Lys Lys       
#                 95                                                      
- aat gat act aaa act aat agt agt agt act ac - #a act aat aat agt agt     
 336                                                                      
Asn Asp Thr Lys Thr Asn Ser Ser Ser Thr Th - #r Thr Asn Asn Ser Ser       
#           110                                                           
- gct aca gct aat agt agt agt act aca act aa - #t agt agt tgg gga gag     
 384                                                                      
Ala Thr Ala Asn Ser Ser Ser Thr Thr Thr As - #n Ser Ser Trp Gly Glu       
#       125                                                               
- ata aag gag gga gaa ata aag aac tgc tct tt - #c aat atc acc aca agc     
 432                                                                      
Ile Lys Glu Gly Glu Ile Lys Asn Cys Ser Ph - #e Asn Ile Thr Thr Ser       
#   140                                                                   
- ata aga gac aag gtg aag aaa gaa tat gca ct - #t ttt tat agc ctt gat     
 480                                                                      
Ile Arg Asp Lys Val Lys Lys Glu Tyr Ala Le - #u Phe Tyr Ser Leu Asp       
145                 1 - #50                 1 - #55                 1 -   
#60                                                                       
- gta gta cca ata gaa aat gat aat act agc ta - #t agg ttg aga agt tgt     
 528                                                                      
Val Val Pro Ile Glu Asn Asp Asn Thr Ser Ty - #r Arg Leu Arg Ser Cys       
#               175                                                       
- aac acc tca gtc att aca caa gcc tgt cca aa - #g gta act ttt gag cca     
 576                                                                      
Asn Thr Ser Val Ile Thr Gln Ala Cys Pro Ly - #s Val Thr Phe Glu Pro       
#           190                                                           
- att ccc ata cat tat tgt acc ccg gct ggt tt - #t gcg att ctg aag tgt     
 624                                                                      
Ile Pro Ile His Tyr Cys Thr Pro Ala Gly Ph - #e Ala Ile Leu Lys Cys       
#       205                                                               
- aga gat aaa aag ttc aat gga aca gga cca tg - #c aaa aat gtt agc aca     
 672                                                                      
Arg Asp Lys Lys Phe Asn Gly Thr Gly Pro Cy - #s Lys Asn Val Ser Thr       
#   220                                                                   
- gta caa tgt gca cat gga att aag cca gta gt - #g tca act caa ctg ctg     
 720                                                                      
Val Gln Cys Ala His Gly Ile Lys Pro Val Va - #l Ser Thr Gln Leu Leu       
225                 2 - #30                 2 - #35                 2 -   
#40                                                                       
- tta aat ggc agc cta gca gaa gaa gag gta at - #a att aga tct gcc aat     
 768                                                                      
Leu Asn Gly Ser Leu Ala Glu Glu Glu Val Il - #e Ile Arg Ser Ala Asn       
#               255                                                       
- ttc tca aac aat gct aaa atc ata ata gta ca - #g ttg agg gaa cct gta     
 816                                                                      
Phe Ser Asn Asn Ala Lys Ile Ile Ile Val Gl - #n Leu Arg Glu Pro Val       
#           270                                                           
- gaa att aat tgt aca aga ccc agc aac aat ac - #a ata aaa ggt ata cac     
 864                                                                      
Glu Ile Asn Cys Thr Arg Pro Ser Asn Asn Th - #r Ile Lys Gly Ile His       
#       285                                                               
- ata gga cca ggg aga gca ttt tat gca aca gg - #a gac ata cga gga gat     
 912                                                                      
Ile Gly Pro Gly Arg Ala Phe Tyr Ala Thr Gl - #y Asp Ile Arg Gly Asp       
#   300                                                                   
- ata aga caa gca cat tgt aac att agt gga gc - #a aaa tgg aat aac act     
 960                                                                      
Ile Arg Gln Ala His Cys Asn Ile Ser Gly Al - #a Lys Trp Asn Asn Thr       
305                 3 - #10                 3 - #15                 3 -   
#20                                                                       
- tta aag aag gta gtt aaa aaa tta aaa gaa ca - #a ttt cca aat aaa aca     
1008                                                                      
Leu Lys Lys Val Val Lys Lys Leu Lys Glu Gl - #n Phe Pro Asn Lys Thr       
#               335                                                       
- ata gtc ttt aac cat tcc tca gga ggg gac cc - #a gaa att gta atg cac     
1056                                                                      
Ile Val Phe Asn His Ser Ser Gly Gly Asp Pr - #o Glu Ile Val Met His       
#           350                                                           
- agt ttt aat tgt caa ggg gaa ttt ttc tac tg - #t aat aca aca aag ctg     
1104                                                                      
Ser Phe Asn Cys Gln Gly Glu Phe Phe Tyr Cy - #s Asn Thr Thr Lys Leu       
#       365                                                               
- ttt aat agt act tgg aat gat act aca gag tc - #a aat aac aat gat agt     
1152                                                                      
Phe Asn Ser Thr Trp Asn Asp Thr Thr Glu Se - #r Asn Asn Asn Asp Ser       
#   380                                                                   
- act att aca ctc cca tgc aga ata aaa caa at - #t ata aac atg tgg cag     
1200                                                                      
Thr Ile Thr Leu Pro Cys Arg Ile Lys Gln Il - #e Ile Asn Met Trp Gln       
385                 3 - #90                 3 - #95                 4 -   
#00                                                                       
- gaa ata gga aaa gca atg tat gcc cct ccc ac - #c aga gga gaa att aaa     
1248                                                                      
Glu Ile Gly Lys Ala Met Tyr Ala Pro Pro Th - #r Arg Gly Glu Ile Lys       
#               415                                                       
- tgt tca tca aat att aca gga cta ctg tta at - #a aga gat ggt ggt att     
1296                                                                      
Cys Ser Ser Asn Ile Thr Gly Leu Leu Leu Il - #e Arg Asp Gly Gly Ile       
#           430                                                           
- aac act agc gat gcc acc gag acc ttc aga cc - #g gga gga gga gat atg     
1344                                                                      
Asn Thr Ser Asp Ala Thr Glu Thr Phe Arg Pr - #o Gly Gly Gly Asp Met       
#       445                                                               
- agg gac aat tgg aga agt gaa tta tat aaa ta - #t aaa gta gtg aaa att     
1392                                                                      
Arg Asp Asn Trp Arg Ser Glu Leu Tyr Lys Ty - #r Lys Val Val Lys Ile       
#   460                                                                   
- gag cca tta gga gta gca ccc acc aag gca aa - #g aga aga gtg gtg cag     
1440                                                                      
Glu Pro Leu Gly Val Ala Pro Thr Lys Ala Ly - #s Arg Arg Val Val Gln       
465                 4 - #70                 4 - #75                 4 -   
#80                                                                       
- aga gaa aaa aga gca gta aca cta gga gct at - #g ttc ctt ggg ttc tta     
1488                                                                      
Arg Glu Lys Arg Ala Val Thr Leu Gly Ala Me - #t Phe Leu Gly Phe Leu       
#               495                                                       
#  1503            tc                                                     
Gly Ala  *  Ser Phe                                                       
            500                                                           
- <210> SEQ ID NO 2                                                       
<211> LENGTH: 498                                                         
<212> TYPE: PRT                                                           
<213> ORGANISM: HIV                                                       
- <400> SEQUENCE: 2                                                       
- Gly Val Pro Val Trp Lys Glu Ala Thr Thr Th - #r Leu Phe Cys Ala Ser     
#                15                                                       
- Asp Ala Lys Ala Tyr Asp Thr Glu Val His As - #n Val Trp Ala Thr His     
#            30                                                           
- Ala Cys Val Pro Thr Asp Pro Asn Pro Gln Gl - #u Met Val Leu Glu Asn     
#        45                                                               
- Val Thr Glu Asp Phe Asn Met Trp Lys Asn As - #p Met Val Glu Gln Met     
#    60                                                                   
- His Glu Asp Ile Ile Ser Leu Trp Asp Gln Se - #r Leu Lys Pro Cys Val     
#80                                                                       
- Lys Leu Thr Pro Leu Cys Ile Thr Leu Asn Cy - #s Thr Asn Trp Lys Lys     
#                95                                                       
- Asn Asp Thr Lys Thr Asn Ser Ser Ser Thr Th - #r Thr Asn Asn Ser Ser     
#           110                                                           
- Ala Thr Ala Asn Ser Ser Ser Thr Thr Thr As - #n Ser Ser Trp Gly Glu     
#       125                                                               
- Ile Lys Glu Gly Glu Ile Lys Asn Cys Ser Ph - #e Asn Ile Thr Thr Ser     
#   140                                                                   
- Ile Arg Asp Lys Val Lys Lys Glu Tyr Ala Le - #u Phe Tyr Ser Leu Asp     
145                 1 - #50                 1 - #55                 1 -   
#60                                                                       
- Val Val Pro Ile Glu Asn Asp Asn Thr Ser Ty - #r Arg Leu Arg Ser Cys     
#               175                                                       
- Asn Thr Ser Val Ile Thr Gln Ala Cys Pro Ly - #s Val Thr Phe Glu Pro     
#           190                                                           
- Ile Pro Ile His Tyr Cys Thr Pro Ala Gly Ph - #e Ala Ile Leu Lys Cys     
#       205                                                               
- Arg Asp Lys Lys Phe Asn Gly Thr Gly Pro Cy - #s Lys Asn Val Ser Thr     
#   220                                                                   
- Val Gln Cys Ala His Gly Ile Lys Pro Val Va - #l Ser Thr Gln Leu Leu     
225                 2 - #30                 2 - #35                 2 -   
#40                                                                       
- Leu Asn Gly Ser Leu Ala Glu Glu Glu Val Il - #e Ile Arg Ser Ala Asn     
#               255                                                       
- Phe Ser Asn Asn Ala Lys Ile Ile Ile Val Gl - #n Leu Arg Glu Pro Val     
#           270                                                           
- Glu Ile Asn Cys Thr Arg Pro Ser Asn Asn Th - #r Ile Lys Gly Ile His     
#       285                                                               
- Ile Gly Pro Gly Arg Ala Phe Tyr Ala Thr Gl - #y Asp Ile Arg Gly Asp     
#   300                                                                   
- Ile Arg Gln Ala His Cys Asn Ile Ser Gly Al - #a Lys Trp Asn Asn Thr     
305                 3 - #10                 3 - #15                 3 -   
#20                                                                       
- Leu Lys Lys Val Val Lys Lys Leu Lys Glu Gl - #n Phe Pro Asn Lys Thr     
#               335                                                       
- Ile Val Phe Asn His Ser Ser Gly Gly Asp Pr - #o Glu Ile Val Met His     
#           350                                                           
- Ser Phe Asn Cys Gln Gly Glu Phe Phe Tyr Cy - #s Asn Thr Thr Lys Leu     
#       365                                                               
- Phe Asn Ser Thr Trp Asn Asp Thr Thr Glu Se - #r Asn Asn Asn Asp Ser     
#   380                                                                   
- Thr Ile Thr Leu Pro Cys Arg Ile Lys Gln Il - #e Ile Asn Met Trp Gln     
385                 3 - #90                 3 - #95                 4 -   
#00                                                                       
- Glu Ile Gly Lys Ala Met Tyr Ala Pro Pro Th - #r Arg Gly Glu Ile Lys     
#               415                                                       
- Cys Ser Ser Asn Ile Thr Gly Leu Leu Leu Il - #e Arg Asp Gly Gly Ile     
#           430                                                           
- Asn Thr Ser Asp Ala Thr Glu Thr Phe Arg Pr - #o Gly Gly Gly Asp Met     
#       445                                                               
- Arg Asp Asn Trp Arg Ser Glu Leu Tyr Lys Ty - #r Lys Val Val Lys Ile     
#   460                                                                   
- Glu Pro Leu Gly Val Ala Pro Thr Lys Ala Ly - #s Arg Arg Val Val Gln     
465                 4 - #70                 4 - #75                 4 -   
#80                                                                       
- Arg Glu Lys Arg Ala Val Thr Leu Gly Ala Me - #t Phe Leu Gly Phe Leu     
#               495                                                       
- Gly Ala                                                                 
- <210> SEQ ID NO 3                                                       
<211> LENGTH: 2                                                           
<212> TYPE: PRT                                                           
<213> ORGANISM: HIV                                                       
- <400> SEQUENCE: 3                                                       
- Ser Phe                                                                 
 1                                                                        
- <210> SEQ ID NO 4                                                       
<211> LENGTH: 1503                                                        
<212> TYPE: DNA                                                           
<213> ORGANISM: HIV                                                       
<220> FEATURE:                                                            
<221> NAME/KEY: CDS                                                       
<222> LOCATION: (1)...(1503)                                              
<221> NAME/KEY: misc.sub.-- feature                                       
<222> LOCATION: (1)...(1503)                                              
<223> OTHER INFORMATION: n = A,T,C or G                                   
- <400> SEQUENCE: 4                                                       
- ggg gta cct gta tgg aaa gaa gca acc acc ac - #t cta ttt tgt gca tca     
  48                                                                      
Gly Val Pro Val Trp Lys Glu Ala Thr Thr Th - #r Leu Phe Cys Ala Ser       
#                 15                                                      
- gat gct aaa gca tat gac aca gag gtg cat aa - #t gtt tgg gcc aca cat     
  96                                                                      
Asp Ala Lys Ala Tyr Asp Thr Glu Val His As - #n Val Trp Ala Thr His       
#             30                                                          
- gct tgt gta ccc aca gac cca aac cca caa ga - #a atg gta ttg gaa aat     
 144                                                                      
Ala Cys Val Pro Thr Asp Pro Asn Pro Gln Gl - #u Met Val Leu Glu Asn       
#         45                                                              
- gtg aca gaa gat ttt aac atg tgg aaa aat ga - #c atg gta gaa cag atg     
 192                                                                      
Val Thr Glu Asp Phe Asn Met Trp Lys Asn As - #p Met Val Glu Gln Met       
#     60                                                                  
- cat gag ant ata atc agt tta tgg gat caa ag - #c cta aaa cca tgt gta     
 240                                                                      
His Glu Xaa Ile Ile Ser Leu Trp Asp Gln Se - #r Leu Lys Pro Cys Val       
# 80                                                                      
- aaa tta acc cca ctc tgt att act tta aat tg - #c acc aat tgg aag gag     
 288                                                                      
Lys Leu Thr Pro Leu Cys Ile Thr Leu Asn Cy - #s Thr Asn Trp Lys Glu       
#                 95                                                      
- aat gat act aaa act aat agt agt agt act ac - #a act aat aat agt agt     
 336                                                                      
Asn Asp Thr Lys Thr Asn Ser Ser Ser Thr Th - #r Thr Asn Asn Ser Ser       
#           110                                                           
- gct aca gct aat agt agt agt act aca act aa - #t agt agt tgg gga gag     
 384                                                                      
Ala Thr Ala Asn Ser Ser Ser Thr Thr Thr As - #n Ser Ser Trp Gly Glu       
#       125                                                               
- ata aag gag gga gaa ata aag aac tgc tct tt - #c aat atc acc aca ggc     
 432                                                                      
Ile Lys Glu Gly Glu Ile Lys Asn Cys Ser Ph - #e Asn Ile Thr Thr Gly       
#   140                                                                   
- ata aga gac aag gtg aag aaa gaa tat gca ct - #t ttt tat agc ctt gat     
 480                                                                      
Ile Arg Asp Lys Val Lys Lys Glu Tyr Ala Le - #u Phe Tyr Ser Leu Asp       
145                 1 - #50                 1 - #55                 1 -   
#60                                                                       
- gta gta cca ata gaa aat gat aat act agc ta - #t agg ttg aga agt tgt     
 528                                                                      
Val Val Pro Ile Glu Asn Asp Asn Thr Ser Ty - #r Arg Leu Arg Ser Cys       
#               175                                                       
- aac acc tca gtc att aca caa gcc tgt cca aa - #g gta act ttt gag cca     
 576                                                                      
Asn Thr Ser Val Ile Thr Gln Ala Cys Pro Ly - #s Val Thr Phe Glu Pro       
#           190                                                           
- att ccc ata cat tat tgt acc ccg gct ggt tt - #t gcg att ctg aag tgt     
 624                                                                      
Ile Pro Ile His Tyr Cys Thr Pro Ala Gly Ph - #e Ala Ile Leu Lys Cys       
#       205                                                               
- aaa gat aaa aag ttc aat gga aca gga cca tg - #c aaa aat gtt agc aca     
 672                                                                      
Lys Asp Lys Lys Phe Asn Gly Thr Gly Pro Cy - #s Lys Asn Val Ser Thr       
#   220                                                                   
- gta caa tgt aca cat gga att aag cca gta gt - #g tca act caa ctg ctg     
 720                                                                      
Val Gln Cys Thr His Gly Ile Lys Pro Val Va - #l Ser Thr Gln Leu Leu       
225                 2 - #30                 2 - #35                 2 -   
#40                                                                       
- tta aat ggc agc cta gca gaa gaa gag gta at - #a att aga tct gcc aat     
 768                                                                      
Leu Asn Gly Ser Leu Ala Glu Glu Glu Val Il - #e Ile Arg Ser Ala Asn       
#               255                                                       
- ttc tca aac aat gct aaa atc ata ata gta ca - #g ttg aag gaa cct gta     
 816                                                                      
Phe Ser Asn Asn Ala Lys Ile Ile Ile Val Gl - #n Leu Lys Glu Pro Val       
#           270                                                           
- gaa att aat tgt aca aga ccc agc aac aat ac - #a ata aaa ggt ata cac     
 864                                                                      
Glu Ile Asn Cys Thr Arg Pro Ser Asn Asn Th - #r Ile Lys Gly Ile His       
#       285                                                               
- ata gga cca ggg aga gca ttt tat gca aca gg - #a gac ata cga gga gat     
 912                                                                      
Ile Gly Pro Gly Arg Ala Phe Tyr Ala Thr Gl - #y Asp Ile Arg Gly Asp       
#   300                                                                   
- ata aga caa gca cat tgt aac att agt gga gc - #a aaa tgg aat aac act     
 960                                                                      
Ile Arg Gln Ala His Cys Asn Ile Ser Gly Al - #a Lys Trp Asn Asn Thr       
305                 3 - #10                 3 - #15                 3 -   
#20                                                                       
- tta aag aag gta gtt ata aaa tta aaa gaa ca - #a ttt cca aat aaa aca     
1008                                                                      
Leu Lys Lys Val Val Ile Lys Leu Lys Glu Gl - #n Phe Pro Asn Lys Thr       
#               335                                                       
- ata gtc ttt aac cat tcc tca gga ggg gac cc - #a gaa att gta atg cac     
1056                                                                      
Ile Val Phe Asn His Ser Ser Gly Gly Asp Pr - #o Glu Ile Val Met His       
#           350                                                           
- agt ttt aat tgt caa ggg gaa ttt ttc tac tg - #t aat aca acg aag ctg     
1104                                                                      
Ser Phe Asn Cys Gln Gly Glu Phe Phe Tyr Cy - #s Asn Thr Thr Lys Leu       
#       365                                                               
- ttt aat agt act tgg aat gat act aca gag tc - #a aat aac aat gat agt     
1152                                                                      
Phe Asn Ser Thr Trp Asn Asp Thr Thr Glu Se - #r Asn Asn Asn Asp Ser       
#   380                                                                   
- act att aca ctc cca tgc aga ata aaa caa at - #t ata aac atg tgg cag     
1200                                                                      
Thr Ile Thr Leu Pro Cys Arg Ile Lys Gln Il - #e Ile Asn Met Trp Gln       
385                 3 - #90                 3 - #95                 4 -   
#00                                                                       
- gaa gta gga aaa gca atg tat gcc cct ccc at - #c aga gga gaa att aaa     
1248                                                                      
Glu Val Gly Lys Ala Met Tyr Ala Pro Pro Il - #e Arg Gly Glu Ile Lys       
#               415                                                       
- tgt tca tca aat att aca gga cta ctg tta ac - #a aga gat ggt ggt att     
1296                                                                      
Cys Ser Ser Asn Ile Thr Gly Leu Leu Leu Th - #r Arg Asp Gly Gly Ile       
#           430                                                           
- aac act agc gat gcc acc gag acc ttc aga cc - #g gga gga gga gat atg     
1344                                                                      
Asn Thr Ser Asp Ala Thr Glu Thr Phe Arg Pr - #o Gly Gly Gly Asp Met       
#       445                                                               
- agg gac aat tgg aga agt gaa tta tat aaa ta - #t aaa gta gtg aaa att     
1392                                                                      
Arg Asp Asn Trp Arg Ser Glu Leu Tyr Lys Ty - #r Lys Val Val Lys Ile       
#   460                                                                   
- gag cca tta gga gta gca ccc acc aag gca aa - #g aga aga gtg gtg cag     
1440                                                                      
Glu Pro Leu Gly Val Ala Pro Thr Lys Ala Ly - #s Arg Arg Val Val Gln       
465                 4 - #70                 4 - #75                 4 -   
#80                                                                       
- aga gaa aaa aga gca gta aca cta gga gct at - #g ttc ctt ggg ttc ttg     
1488                                                                      
Arg Glu Lys Arg Ala Val Thr Leu Gly Ala Me - #t Phe Leu Gly Phe Leu       
#               495                                                       
#  1503            tc                                                     
Gly Ala  *  Ser Phe                                                       
            500                                                           
- <210> SEQ ID NO 5                                                       
<211> LENGTH: 498                                                         
<212> TYPE: PRT                                                           
<213> ORGANISM: HIV                                                       
<220> FEATURE:                                                            
<221> NAME/KEY: VARIANT                                                   
<222> LOCATION: (1)...(498)                                               
<223> OTHER INFORMATION: Xaa = Any Amino Aci - #d                         
- <400> SEQUENCE: 5                                                       
- Gly Val Pro Val Trp Lys Glu Ala Thr Thr Th - #r Leu Phe Cys Ala Ser     
#                15                                                       
- Asp Ala Lys Ala Tyr Asp Thr Glu Val His As - #n Val Trp Ala Thr His     
#            30                                                           
- Ala Cys Val Pro Thr Asp Pro Asn Pro Gln Gl - #u Met Val Leu Glu Asn     
#        45                                                               
- Val Thr Glu Asp Phe Asn Met Trp Lys Asn As - #p Met Val Glu Gln Met     
#    60                                                                   
- His Glu Xaa Ile Ile Ser Leu Trp Asp Gln Se - #r Leu Lys Pro Cys Val     
#80                                                                       
- Lys Leu Thr Pro Leu Cys Ile Thr Leu Asn Cy - #s Thr Asn Trp Lys Glu     
#                95                                                       
- Asn Asp Thr Lys Thr Asn Ser Ser Ser Thr Th - #r Thr Asn Asn Ser Ser     
#           110                                                           
- Ala Thr Ala Asn Ser Ser Ser Thr Thr Thr As - #n Ser Ser Trp Gly Glu     
#       125                                                               
- Ile Lys Glu Gly Glu Ile Lys Asn Cys Ser Ph - #e Asn Ile Thr Thr Gly     
#   140                                                                   
- Ile Arg Asp Lys Val Lys Lys Glu Tyr Ala Le - #u Phe Tyr Ser Leu Asp     
145                 1 - #50                 1 - #55                 1 -   
#60                                                                       
- Val Val Pro Ile Glu Asn Asp Asn Thr Ser Ty - #r Arg Leu Arg Ser Cys     
#               175                                                       
- Asn Thr Ser Val Ile Thr Gln Ala Cys Pro Ly - #s Val Thr Phe Glu Pro     
#           190                                                           
- Ile Pro Ile His Tyr Cys Thr Pro Ala Gly Ph - #e Ala Ile Leu Lys Cys     
#       205                                                               
- Lys Asp Lys Lys Phe Asn Gly Thr Gly Pro Cy - #s Lys Asn Val Ser Thr     
#   220                                                                   
- Val Gln Cys Thr His Gly Ile Lys Pro Val Va - #l Ser Thr Gln Leu Leu     
225                 2 - #30                 2 - #35                 2 -   
#40                                                                       
- Leu Asn Gly Ser Leu Ala Glu Glu Glu Val Il - #e Ile Arg Ser Ala Asn     
#               255                                                       
- Phe Ser Asn Asn Ala Lys Ile Ile Ile Val Gl - #n Leu Lys Glu Pro Val     
#           270                                                           
- Glu Ile Asn Cys Thr Arg Pro Ser Asn Asn Th - #r Ile Lys Gly Ile His     
#       285                                                               
- Ile Gly Pro Gly Arg Ala Phe Tyr Ala Thr Gl - #y Asp Ile Arg Gly Asp     
#   300                                                                   
- Ile Arg Gln Ala His Cys Asn Ile Ser Gly Al - #a Lys Trp Asn Asn Thr     
305                 3 - #10                 3 - #15                 3 -   
#20                                                                       
- Leu Lys Lys Val Val Ile Lys Leu Lys Glu Gl - #n Phe Pro Asn Lys Thr     
#               335                                                       
- Ile Val Phe Asn His Ser Ser Gly Gly Asp Pr - #o Glu Ile Val Met His     
#           350                                                           
- Ser Phe Asn Cys Gln Gly Glu Phe Phe Tyr Cy - #s Asn Thr Thr Lys Leu     
#       365                                                               
- Phe Asn Ser Thr Trp Asn Asp Thr Thr Glu Se - #r Asn Asn Asn Asp Ser     
#   380                                                                   
- Thr Ile Thr Leu Pro Cys Arg Ile Lys Gln Il - #e Ile Asn Met Trp Gln     
385                 3 - #90                 3 - #95                 4 -   
#00                                                                       
- Glu Val Gly Lys Ala Met Tyr Ala Pro Pro Il - #e Arg Gly Glu Ile Lys     
#               415                                                       
- Cys Ser Ser Asn Ile Thr Gly Leu Leu Leu Th - #r Arg Asp Gly Gly Ile     
#           430                                                           
- Asn Thr Ser Asp Ala Thr Glu Thr Phe Arg Pr - #o Gly Gly Gly Asp Met     
#       445                                                               
- Arg Asp Asn Trp Arg Ser Glu Leu Tyr Lys Ty - #r Lys Val Val Lys Ile     
#   460                                                                   
- Glu Pro Leu Gly Val Ala Pro Thr Lys Ala Ly - #s Arg Arg Val Val Gln     
465                 4 - #70                 4 - #75                 4 -   
#80                                                                       
- Arg Glu Lys Arg Ala Val Thr Leu Gly Ala Me - #t Phe Leu Gly Phe Leu     
#               495                                                       
- Gly Ala                                                                 
- <210> SEQ ID NO 6                                                       
<211> LENGTH: 2                                                           
<212> TYPE: PRT                                                           
<213> ORGANISM: HIV                                                       
- <400> SEQUENCE: 6                                                       
- Ser Phe                                                                 
 1                                                                        
- <210> SEQ ID NO 7                                                       
<211> LENGTH: 1461                                                        
<212> TYPE: DNA                                                           
<213> ORGANISM: HIV                                                       
<220> FEATURE:                                                            
<221> NAME/KEY: CDS                                                       
<222> LOCATION: (2)...(1459)                                              
- <400> SEQUENCE: 7                                                       
#cta ttt tgt gca tca gat     49 acc acc act                               
  Val Pro Val Trp Lys Glu Ala Thr Thr T - #hr Leu Phe Cys Ala Ser Asp     
# 15                                                                      
- gct aaa gca tat gat aca gag gta cat aat gt - #t tgg gct aca cat gcc     
  97                                                                      
Ala Lys Ala Tyr Asp Thr Glu Val His Asn Va - #l Trp Ala Thr His Ala       
#             30                                                          
- tgt gta ccc aca gac ccc aac cca caa gaa gt - #a gta ttg gaa aat gta     
 145                                                                      
Cys Val Pro Thr Asp Pro Asn Pro Gln Glu Va - #l Val Leu Glu Asn Val       
#         45                                                              
- aca gaa aat ttt aac atg tgg aaa aat aac at - #g gta gaa cag atg cat     
 193                                                                      
Thr Glu Asn Phe Asn Met Trp Lys Asn Asn Me - #t Val Glu Gln Met His       
#     60                                                                  
- gag gat ata atc agt tta tgg gat caa agt ct - #a aag cca tgt gta aaa     
 241                                                                      
Glu Asp Ile Ile Ser Leu Trp Asp Gln Ser Le - #u Lys Pro Cys Val Lys       
# 80                                                                      
- tta acc cca ctc tgt gtt act tta aat tgc ac - #t aat ttg gag aat gct     
 289                                                                      
Leu Thr Pro Leu Cys Val Thr Leu Asn Cys Th - #r Asn Leu Glu Asn Ala       
#                 95                                                      
- aat aat acc gag aat gct aat aat acc aat aa - #t tat acc ttg ggg atg     
 337                                                                      
Asn Asn Thr Glu Asn Ala Asn Asn Thr Asn As - #n Tyr Thr Leu Gly Met       
#           110                                                           
- gag aga ggt gaa ata aaa aac tgc tct ttc aa - #t atc acc aca agc tta     
 385                                                                      
Glu Arg Gly Glu Ile Lys Asn Cys Ser Phe As - #n Ile Thr Thr Ser Leu       
#       125                                                               
- aga gat aag gtg aaa aaa gaa tat gca ttg tt - #t tat aaa ctt gat gta     
 433                                                                      
Arg Asp Lys Val Lys Lys Glu Tyr Ala Leu Ph - #e Tyr Lys Leu Asp Val       
#   140                                                                   
- gta caa ata gat aat agt acc aac tat agg ct - #g ata agt tgt aat acc     
 481                                                                      
Val Gln Ile Asp Asn Ser Thr Asn Tyr Arg Le - #u Ile Ser Cys Asn Thr       
145                 1 - #50                 1 - #55                 1 -   
#60                                                                       
- tca gtc att aca cag gcc tgt cca aag gta tc - #c ttt gag cta att ccc     
 529                                                                      
Ser Val Ile Thr Gln Ala Cys Pro Lys Val Se - #r Phe Glu Leu Ile Pro       
#               175                                                       
- ata cat tat tgt gcc ccg gct ggt ttt gcg at - #t cta aag tgt aaa gat     
 577                                                                      
Ile His Tyr Cys Ala Pro Ala Gly Phe Ala Il - #e Leu Lys Cys Lys Asp       
#           190                                                           
- aag aag ttc aat gga aca gga cca tgt aaa aa - #t gtc agc aca gta caa     
 625                                                                      
Lys Lys Phe Asn Gly Thr Gly Pro Cys Lys As - #n Val Ser Thr Val Gln       
#       205                                                               
- tgt aca cat gga att aga cca gta gta tca ac - #t caa cta ctg tta aat     
 673                                                                      
Cys Thr His Gly Ile Arg Pro Val Val Ser Th - #r Gln Leu Leu Leu Asn       
#   220                                                                   
- ggc agt cta gca gaa gaa gag ata gta att ag - #a tct gaa aat atc aca     
 721                                                                      
Gly Ser Leu Ala Glu Glu Glu Ile Val Ile Ar - #g Ser Glu Asn Ile Thr       
225                 2 - #30                 2 - #35                 2 -   
#40                                                                       
- gac aat gct aaa acc ata ata gtg cag cta aa - #t gaa tct ata gtg att     
 769                                                                      
Asp Asn Ala Lys Thr Ile Ile Val Gln Leu As - #n Glu Ser Ile Val Ile       
#               255                                                       
- aat tgt aca aga ccc aat aac aac aca aga aa - #a agt ata aat ata gga     
 817                                                                      
Asn Cys Thr Arg Pro Asn Asn Asn Thr Arg Ly - #s Ser Ile Asn Ile Gly       
#           270                                                           
- cca ggg aga gca ttc tat aca aca gga gac at - #a ata gga gat ata aga     
 865                                                                      
Pro Gly Arg Ala Phe Tyr Thr Thr Gly Asp Il - #e Ile Gly Asp Ile Arg       
#       285                                                               
- caa gca cat tgt aac ctt agt aaa aca caa tg - #g gaa aaa acg tta aga     
 913                                                                      
Gln Ala His Cys Asn Leu Ser Lys Thr Gln Tr - #p Glu Lys Thr Leu Arg       
#   300                                                                   
- cag ata gct ata aaa tta gaa gaa aaa ttt aa - #g aat aaa aca ata gcc     
 961                                                                      
Gln Ile Ala Ile Lys Leu Glu Glu Lys Phe Ly - #s Asn Lys Thr Ile Ala       
305                 3 - #10                 3 - #15                 3 -   
#20                                                                       
- ttt aat aaa tcc tca gga ggg gac cca gaa at - #t gta atg cac agt ttt     
1009                                                                      
Phe Asn Lys Ser Ser Gly Gly Asp Pro Glu Il - #e Val Met His Ser Phe       
#               335                                                       
- aat tgt gga ggg gaa ttt ttc tac tgt aat ac - #a aca aaa ctg ttt aat     
1057                                                                      
Asn Cys Gly Gly Glu Phe Phe Tyr Cys Asn Th - #r Thr Lys Leu Phe Asn       
#           350                                                           
- agt acc tgg aat tta aca caa ccg ttt agt aa - #t acc ggg aat cgt act     
1105                                                                      
Ser Thr Trp Asn Leu Thr Gln Pro Phe Ser As - #n Thr Gly Asn Arg Thr       
#       365                                                               
- gaa gag tta aat att aca ctc cca tgc aga at - #a aaa caa atc ata aac     
1153                                                                      
Glu Glu Leu Asn Ile Thr Leu Pro Cys Arg Il - #e Lys Gln Ile Ile Asn       
#   380                                                                   
- ttg tgg cag gaa gta ggc aaa gca atg tat gc - #c cct ccc atc aga gga     
1201                                                                      
Leu Trp Gln Glu Val Gly Lys Ala Met Tyr Al - #a Pro Pro Ile Arg Gly       
385                 3 - #90                 3 - #95                 4 -   
#00                                                                       
- caa att aga tgt tca tca aat att aca ggg ct - #a cta tta aca aga gat     
1249                                                                      
Gln Ile Arg Cys Ser Ser Asn Ile Thr Gly Le - #u Leu Leu Thr Arg Asp       
#               415                                                       
- ggt gga agt aac acc ggt gac aac agg act ga - #g acc ttt aga cct gga     
1297                                                                      
Gly Gly Ser Asn Thr Gly Asp Asn Arg Thr Gl - #u Thr Phe Arg Pro Gly       
#           430                                                           
- gga gga gat atg agg gac aat tgg aga agt ga - #a tta tat aaa tat aaa     
1345                                                                      
Gly Gly Asp Met Arg Asp Asn Trp Arg Ser Gl - #u Leu Tyr Lys Tyr Lys       
#       445                                                               
- gta gta aga att gaa cca tta gga gta gca cc - #c acc cag gca aag aga     
1393                                                                      
Val Val Arg Ile Glu Pro Leu Gly Val Ala Pr - #o Thr Gln Ala Lys Arg       
#   460                                                                   
- aga gtg gtg caa aga gaa aaa aga gca gtg gg - #g ata gga gct atg ttc     
1441                                                                      
Arg Val Val Gln Arg Glu Lys Arg Ala Val Gl - #y Ile Gly Ala Met Phe       
465                 4 - #70                 4 - #75                 4 -   
#80                                                                       
#                 146 - #1aa                                              
Leu Gly Phe Leu Gly Asp                                                   
                485                                                       
- <210> SEQ ID NO 8                                                       
<211> LENGTH: 486                                                         
<212> TYPE: PRT                                                           
<213> ORGANISM: HIV                                                       
- <400> SEQUENCE: 8                                                       
- Val Pro Val Trp Lys Glu Ala Thr Thr Thr Le - #u Phe Cys Ala Ser Asp     
#                15                                                       
- Ala Lys Ala Tyr Asp Thr Glu Val His Asn Va - #l Trp Ala Thr His Ala     
#            30                                                           
- Cys Val Pro Thr Asp Pro Asn Pro Gln Glu Va - #l Val Leu Glu Asn Val     
#        45                                                               
- Thr Glu Asn Phe Asn Met Trp Lys Asn Asn Me - #t Val Glu Gln Met His     
#    60                                                                   
- Glu Asp Ile Ile Ser Leu Trp Asp Gln Ser Le - #u Lys Pro Cys Val Lys     
#80                                                                       
- Leu Thr Pro Leu Cys Val Thr Leu Asn Cys Th - #r Asn Leu Glu Asn Ala     
#                95                                                       
- Asn Asn Thr Glu Asn Ala Asn Asn Thr Asn As - #n Tyr Thr Leu Gly Met     
#           110                                                           
- Glu Arg Gly Glu Ile Lys Asn Cys Ser Phe As - #n Ile Thr Thr Ser Leu     
#       125                                                               
- Arg Asp Lys Val Lys Lys Glu Tyr Ala Leu Ph - #e Tyr Lys Leu Asp Val     
#   140                                                                   
- Val Gln Ile Asp Asn Ser Thr Asn Tyr Arg Le - #u Ile Ser Cys Asn Thr     
145                 1 - #50                 1 - #55                 1 -   
#60                                                                       
- Ser Val Ile Thr Gln Ala Cys Pro Lys Val Se - #r Phe Glu Leu Ile Pro     
#               175                                                       
- Ile His Tyr Cys Ala Pro Ala Gly Phe Ala Il - #e Leu Lys Cys Lys Asp     
#           190                                                           
- Lys Lys Phe Asn Gly Thr Gly Pro Cys Lys As - #n Val Ser Thr Val Gln     
#       205                                                               
- Cys Thr His Gly Ile Arg Pro Val Val Ser Th - #r Gln Leu Leu Leu Asn     
#   220                                                                   
- Gly Ser Leu Ala Glu Glu Glu Ile Val Ile Ar - #g Ser Glu Asn Ile Thr     
225                 2 - #30                 2 - #35                 2 -   
#40                                                                       
- Asp Asn Ala Lys Thr Ile Ile Val Gln Leu As - #n Glu Ser Ile Val Ile     
#               255                                                       
- Asn Cys Thr Arg Pro Asn Asn Asn Thr Arg Ly - #s Ser Ile Asn Ile Gly     
#           270                                                           
- Pro Gly Arg Ala Phe Tyr Thr Thr Gly Asp Il - #e Ile Gly Asp Ile Arg     
#       285                                                               
- Gln Ala His Cys Asn Leu Ser Lys Thr Gln Tr - #p Glu Lys Thr Leu Arg     
#   300                                                                   
- Gln Ile Ala Ile Lys Leu Glu Glu Lys Phe Ly - #s Asn Lys Thr Ile Ala     
305                 3 - #10                 3 - #15                 3 -   
#20                                                                       
- Phe Asn Lys Ser Ser Gly Gly Asp Pro Glu Il - #e Val Met His Ser Phe     
#               335                                                       
- Asn Cys Gly Gly Glu Phe Phe Tyr Cys Asn Th - #r Thr Lys Leu Phe Asn     
#           350                                                           
- Ser Thr Trp Asn Leu Thr Gln Pro Phe Ser As - #n Thr Gly Asn Arg Thr     
#       365                                                               
- Glu Glu Leu Asn Ile Thr Leu Pro Cys Arg Il - #e Lys Gln Ile Ile Asn     
#   380                                                                   
- Leu Trp Gln Glu Val Gly Lys Ala Met Tyr Al - #a Pro Pro Ile Arg Gly     
385                 3 - #90                 3 - #95                 4 -   
#00                                                                       
- Gln Ile Arg Cys Ser Ser Asn Ile Thr Gly Le - #u Leu Leu Thr Arg Asp     
#               415                                                       
- Gly Gly Ser Asn Thr Gly Asp Asn Arg Thr Gl - #u Thr Phe Arg Pro Gly     
#           430                                                           
- Gly Gly Asp Met Arg Asp Asn Trp Arg Ser Gl - #u Leu Tyr Lys Tyr Lys     
#       445                                                               
- Val Val Arg Ile Glu Pro Leu Gly Val Ala Pr - #o Thr Gln Ala Lys Arg     
#   460                                                                   
- Arg Val Val Gln Arg Glu Lys Arg Ala Val Gl - #y Ile Gly Ala Met Phe     
465                 4 - #70                 4 - #75                 4 -   
#80                                                                       
- Leu Gly Phe Leu Gly Asp                                                 
                485                                                       
- <210> SEQ ID NO 9                                                       
<211> LENGTH: 1474                                                        
<212> TYPE: DNA                                                           
<213> ORGANISM: HIV                                                       
<220> FEATURE:                                                            
<221> NAME/KEY: CDS                                                       
<222> LOCATION: (2)...(1474)                                              
- <400> SEQUENCE: 9                                                       
#cta ttt tgt gca tca gat     49 acc acc act                               
  Val Pro Val Trp Lys Glu Ala Thr Thr T - #hr Leu Phe Cys Ala Ser Asp     
# 15                                                                      
- gct aaa gca tat gat aca gag gta cat aat gt - #t tgg gct aca cat gcc     
  97                                                                      
Ala Lys Ala Tyr Asp Thr Glu Val His Asn Va - #l Trp Ala Thr His Ala       
#             30                                                          
- tgt gta ccc aca gac ccc aac cca caa gaa gt - #a gta ttg gaa aat gta     
 145                                                                      
Cys Val Pro Thr Asp Pro Asn Pro Gln Glu Va - #l Val Leu Glu Asn Val       
#         45                                                              
- aca gaa aat ttt aac atg tgg aaa aat aac at - #g gta gaa cag atg cat     
 193                                                                      
Thr Glu Asn Phe Asn Met Trp Lys Asn Asn Me - #t Val Glu Gln Met His       
#     60                                                                  
- gag gat ata atc agt tta tgg gat caa agt ct - #a aag cca tgt gta aaa     
 241                                                                      
Glu Asp Ile Ile Ser Leu Trp Asp Gln Ser Le - #u Lys Pro Cys Val Lys       
# 80                                                                      
- tta acc cca ctc tgt gtt act tta aat tgc ac - #t aat ttg gag aat gct     
 289                                                                      
Leu Thr Pro Leu Cys Val Thr Leu Asn Cys Th - #r Asn Leu Glu Asn Ala       
#                 95                                                      
- aat aat acc gag aat gct aat aat acc aat aa - #t tat acc ttg ggg atg     
 337                                                                      
Asn Asn Thr Glu Asn Ala Asn Asn Thr Asn As - #n Tyr Thr Leu Gly Met       
#           110                                                           
- gag aga ggt gaa aga aaa aac tgc tct ttc aa - #t atc acc aca agc tta     
 385                                                                      
Glu Arg Gly Glu Arg Lys Asn Cys Ser Phe As - #n Ile Thr Thr Ser Leu       
#       125                                                               
- aga gat aag ggg aaa aaa gaa tat gca ttg tt - #t tat aaa ctt gat gta     
 433                                                                      
Arg Asp Lys Gly Lys Lys Glu Tyr Ala Leu Ph - #e Tyr Lys Leu Asp Val       
#   140                                                                   
- gta caa ata gat aat agt acc aac tat agg ct - #g ata agt tgt aat acc     
 481                                                                      
Val Gln Ile Asp Asn Ser Thr Asn Tyr Arg Le - #u Ile Ser Cys Asn Thr       
145                 1 - #50                 1 - #55                 1 -   
#60                                                                       
- tca gtc att aca cag gcc tgt cca aag gta tc - #c ttt gag cca att ccc     
 529                                                                      
Ser Val Ile Thr Gln Ala Cys Pro Lys Val Se - #r Phe Glu Pro Ile Pro       
#               175                                                       
- ata cat tat tgt gcc ccg gct ggt ttt gcg at - #t cta aag tgt aaa gat     
 577                                                                      
Ile His Tyr Cys Ala Pro Ala Gly Phe Ala Il - #e Leu Lys Cys Lys Asp       
#           190                                                           
- aag aag ttc aat gga aca gga cca tgt aaa aa - #t gtc agg aca gta caa     
 625                                                                      
Lys Lys Phe Asn Gly Thr Gly Pro Cys Lys As - #n Val Arg Thr Val Gln       
#       205                                                               
- tgt aca cat gga att aga cca gta gta tca ac - #t caa cta ctg tta aat     
 673                                                                      
Cys Thr His Gly Ile Arg Pro Val Val Ser Th - #r Gln Leu Leu Leu Asn       
#   220                                                                   
- ggc agt cta gca gaa gaa gag ata gta att ag - #a tct gaa aat atc aca     
 721                                                                      
Gly Ser Leu Ala Glu Glu Glu Ile Val Ile Ar - #g Ser Glu Asn Ile Thr       
225                 2 - #30                 2 - #35                 2 -   
#40                                                                       
- gac aat gct aaa acc ata ata gtg cag cta aa - #t gaa tct ata gtg att     
 769                                                                      
Asp Asn Ala Lys Thr Ile Ile Val Gln Leu As - #n Glu Ser Ile Val Ile       
#               255                                                       
- aat tgt aca aga ccc aat aac aac aca aga aa - #a agt ata aat ata gga     
 817                                                                      
Asn Cys Thr Arg Pro Asn Asn Asn Thr Arg Ly - #s Ser Ile Asn Ile Gly       
#           270                                                           
- cca ggg aga gca ttc tat aca aca gga gac at - #a ata gga gat ata aga     
 865                                                                      
Pro Gly Arg Ala Phe Tyr Thr Thr Gly Asp Il - #e Ile Gly Asp Ile Arg       
#       285                                                               
- caa gca cat tgt aac ctt agt aaa aca caa tg - #g gaa aaa acg tta aga     
 913                                                                      
Gln Ala His Cys Asn Leu Ser Lys Thr Gln Tr - #p Glu Lys Thr Leu Arg       
#   300                                                                   
- cag ata gct ata aaa tta gaa gaa aaa ttt aa - #g aat aaa aca ata gcc     
 961                                                                      
Gln Ile Ala Ile Lys Leu Glu Glu Lys Phe Ly - #s Asn Lys Thr Ile Ala       
305                 3 - #10                 3 - #15                 3 -   
#20                                                                       
- ttt aat aaa tcc tca gga ggg gac cca gaa at - #t gta atg cac agt ttt     
1009                                                                      
Phe Asn Lys Ser Ser Gly Gly Asp Pro Glu Il - #e Val Met His Ser Phe       
#               335                                                       
- aat tgt gga ggg gga ttt ttc tac tgt agt ac - #g aga aaa ctg ttt aat     
1057                                                                      
Asn Cys Gly Gly Gly Phe Phe Tyr Cys Ser Th - #r Arg Lys Leu Phe Asn       
#           350                                                           
- agt acc tgg aat tta aca caa ccg ttt agt aa - #t acc ggg gat cgt act     
1105                                                                      
Ser Thr Trp Asn Leu Thr Gln Pro Phe Ser As - #n Thr Gly Asp Arg Thr       
#       365                                                               
- gaa gag tta aat att aca ctc cca tgc aga at - #a aaa caa atc ata aac     
1153                                                                      
Glu Glu Leu Asn Ile Thr Leu Pro Cys Arg Il - #e Lys Gln Ile Ile Asn       
#   380                                                                   
- ttg tgg cag gaa gta ggc aaa gca atg tat gc - #c cct ccc atc aga gga     
1201                                                                      
Leu Trp Gln Glu Val Gly Lys Ala Met Tyr Al - #a Pro Pro Ile Arg Gly       
385                 3 - #90                 3 - #95                 4 -   
#00                                                                       
- caa att aga tgt tca tca aat att aca ggg ct - #a cta tta agg aga gat     
1249                                                                      
Gln Ile Arg Cys Ser Ser Asn Ile Thr Gly Le - #u Leu Leu Arg Arg Asp       
#               415                                                       
- ggt gga agt aac acc agt gac aac cag act ga - #g acc ttt aga cct ggg     
1297                                                                      
Gly Gly Ser Asn Thr Ser Asp Asn Gln Thr Gl - #u Thr Phe Arg Pro Gly       
#           430                                                           
- gga gga gat atg agg gac aag tgg aga agt ga - #a tta tat aaa tat aaa     
1345                                                                      
Gly Gly Asp Met Arg Asp Lys Trp Arg Ser Gl - #u Leu Tyr Lys Tyr Lys       
#       445                                                               
- gta gta aga att gaa cca tta gga gta gca cc - #c acc cag gca aag aga     
1393                                                                      
Val Val Arg Ile Glu Pro Leu Gly Val Ala Pr - #o Thr Gln Ala Lys Arg       
#   460                                                                   
- aga gtg gtg caa aga gaa aaa aga gca gtg gg - #g ata gga gct atg ttc     
1441                                                                      
Arg Val Val Gln Arg Glu Lys Arg Ala Val Gl - #y Ile Gly Ala Met Phe       
465                 4 - #70                 4 - #75                 4 -   
#80                                                                       
#       1474c tta gga gat aaa gct tct aga gt - #c                         
Leu Arg Phe Leu Gly Asp Lys Ala Ser Arg Va - #l                           
#               490                                                       
- <210> SEQ ID NO 10                                                      
<211> LENGTH: 491                                                         
<212> TYPE: PRT                                                           
<213> ORGANISM: HIV                                                       
- <400> SEQUENCE: 10                                                      
- Val Pro Val Trp Lys Glu Ala Thr Thr Thr Le - #u Phe Cys Ala Ser Asp     
#                15                                                       
- Ala Lys Ala Tyr Asp Thr Glu Val His Asn Va - #l Trp Ala Thr His Ala     
#            30                                                           
- Cys Val Pro Thr Asp Pro Asn Pro Gln Glu Va - #l Val Leu Glu Asn Val     
#        45                                                               
- Thr Glu Asn Phe Asn Met Trp Lys Asn Asn Me - #t Val Glu Gln Met His     
#    60                                                                   
- Glu Asp Ile Ile Ser Leu Trp Asp Gln Ser Le - #u Lys Pro Cys Val Lys     
#80                                                                       
- Leu Thr Pro Leu Cys Val Thr Leu Asn Cys Th - #r Asn Leu Glu Asn Ala     
#                95                                                       
- Asn Asn Thr Glu Asn Ala Asn Asn Thr Asn As - #n Tyr Thr Leu Gly Met     
#           110                                                           
- Glu Arg Gly Glu Arg Lys Asn Cys Ser Phe As - #n Ile Thr Thr Ser Leu     
#       125                                                               
- Arg Asp Lys Gly Lys Lys Glu Tyr Ala Leu Ph - #e Tyr Lys Leu Asp Val     
#   140                                                                   
- Val Gln Ile Asp Asn Ser Thr Asn Tyr Arg Le - #u Ile Ser Cys Asn Thr     
145                 1 - #50                 1 - #55                 1 -   
#60                                                                       
- Ser Val Ile Thr Gln Ala Cys Pro Lys Val Se - #r Phe Glu Pro Ile Pro     
#               175                                                       
- Ile His Tyr Cys Ala Pro Ala Gly Phe Ala Il - #e Leu Lys Cys Lys Asp     
#           190                                                           
- Lys Lys Phe Asn Gly Thr Gly Pro Cys Lys As - #n Val Arg Thr Val Gln     
#       205                                                               
- Cys Thr His Gly Ile Arg Pro Val Val Ser Th - #r Gln Leu Leu Leu Asn     
#   220                                                                   
- Gly Ser Leu Ala Glu Glu Glu Ile Val Ile Ar - #g Ser Glu Asn Ile Thr     
225                 2 - #30                 2 - #35                 2 -   
#40                                                                       
- Asp Asn Ala Lys Thr Ile Ile Val Gln Leu As - #n Glu Ser Ile Val Ile     
#               255                                                       
- Asn Cys Thr Arg Pro Asn Asn Asn Thr Arg Ly - #s Ser Ile Asn Ile Gly     
#           270                                                           
- Pro Gly Arg Ala Phe Tyr Thr Thr Gly Asp Il - #e Ile Gly Asp Ile Arg     
#       285                                                               
- Gln Ala His Cys Asn Leu Ser Lys Thr Gln Tr - #p Glu Lys Thr Leu Arg     
#   300                                                                   
- Gln Ile Ala Ile Lys Leu Glu Glu Lys Phe Ly - #s Asn Lys Thr Ile Ala     
305                 3 - #10                 3 - #15                 3 -   
#20                                                                       
- Phe Asn Lys Ser Ser Gly Gly Asp Pro Glu Il - #e Val Met His Ser Phe     
#               335                                                       
- Asn Cys Gly Gly Gly Phe Phe Tyr Cys Ser Th - #r Arg Lys Leu Phe Asn     
#           350                                                           
- Ser Thr Trp Asn Leu Thr Gln Pro Phe Ser As - #n Thr Gly Asp Arg Thr     
#       365                                                               
- Glu Glu Leu Asn Ile Thr Leu Pro Cys Arg Il - #e Lys Gln Ile Ile Asn     
#   380                                                                   
- Leu Trp Gln Glu Val Gly Lys Ala Met Tyr Al - #a Pro Pro Ile Arg Gly     
385                 3 - #90                 3 - #95                 4 -   
#00                                                                       
- Gln Ile Arg Cys Ser Ser Asn Ile Thr Gly Le - #u Leu Leu Arg Arg Asp     
#               415                                                       
- Gly Gly Ser Asn Thr Ser Asp Asn Gln Thr Gl - #u Thr Phe Arg Pro Gly     
#           430                                                           
- Gly Gly Asp Met Arg Asp Lys Trp Arg Ser Gl - #u Leu Tyr Lys Tyr Lys     
#       445                                                               
- Val Val Arg Ile Glu Pro Leu Gly Val Ala Pr - #o Thr Gln Ala Lys Arg     
#   460                                                                   
- Arg Val Val Gln Arg Glu Lys Arg Ala Val Gl - #y Ile Gly Ala Met Phe     
465                 4 - #70                 4 - #75                 4 -   
#80                                                                       
- Leu Arg Phe Leu Gly Asp Lys Ala Ser Arg Va - #l                         
#               490                                                       
- <210> SEQ ID NO 11                                                      
<211> LENGTH: 1512                                                        
<212> TYPE: DNA                                                           
<213> ORGANISM: HIV                                                       
<220> FEATURE:                                                            
<221> NAME/KEY: CDS                                                       
<222> LOCATION: (1)...(1512)                                              
- <400> SEQUENCE: 11                                                      
- ctc gag gta cct gta tgg aaa gaa gca act ac - #c act cta ttt tgt gca     
  48                                                                      
Leu Glu Val Pro Val Trp Lys Glu Ala Thr Th - #r Thr Leu Phe Cys Ala       
#                 15                                                      
- tca gat gct aaa gca tat aat aca gag aaa ca - #t aat gtt tgg gcc aca     
  96                                                                      
Ser Asp Ala Lys Ala Tyr Asn Thr Glu Lys Hi - #s Asn Val Trp Ala Thr       
#             30                                                          
- cac gcc tgt gta ccc aca gat ccc aac cca ca - #a gaa gta gta ttg gga     
 144                                                                      
His Ala Cys Val Pro Thr Asp Pro Asn Pro Gl - #n Glu Val Val Leu Gly       
#         45                                                              
- aat gtg aca gaa aat ttt aac atg tgg aaa aa - #t aac atg gta gaa caa     
 192                                                                      
Asn Val Thr Glu Asn Phe Asn Met Trp Lys As - #n Asn Met Val Glu Gln       
#     60                                                                  
- atg cat gaa gat ata atc agt tta tgg gat ca - #a agt cta aag cca tgt     
 240                                                                      
Met His Glu Asp Ile Ile Ser Leu Trp Asp Gl - #n Ser Leu Lys Pro Cys       
# 80                                                                      
- gta aaa tta acc cca ctc tgt gtt act tta aa - #t tgc act gat gat tta     
 288                                                                      
Val Lys Leu Thr Pro Leu Cys Val Thr Leu As - #n Cys Thr Asp Asp Leu       
#                 95                                                      
- ggg aat gct act aat acc aat agt agt gcc ac - #t acc aat agt agt agt     
 336                                                                      
Gly Asn Ala Thr Asn Thr Asn Ser Ser Ala Th - #r Thr Asn Ser Ser Ser       
#           110                                                           
- tgg gaa gaa atg aag ggg gaa atg aaa aga tg - #c tct ttc aat atc acc     
 384                                                                      
Trp Glu Glu Met Lys Gly Glu Met Lys Arg Cy - #s Ser Phe Asn Ile Thr       
#       125                                                               
- aca agc ata aga gat aag att aag aaa gaa ca - #t gca ctt ttc tat aga     
 432                                                                      
Thr Ser Ile Arg Asp Lys Ile Lys Lys Glu Hi - #s Ala Leu Phe Tyr Arg       
#   140                                                                   
- ctt gat gta gta cca ata gat aat gat aat ac - #c aca tat agg ttg ata     
 480                                                                      
Leu Asp Val Val Pro Ile Asp Asn Asp Asn Th - #r Thr Tyr Arg Leu Ile       
145                 1 - #50                 1 - #55                 1 -   
#60                                                                       
- aat tgt aat acc tca gtc att aca cag gcc tg - #t cca aag gta tca ttt     
 528                                                                      
Asn Cys Asn Thr Ser Val Ile Thr Gln Ala Cy - #s Pro Lys Val Ser Phe       
#               175                                                       
- gag cca att ccc ata cat ttt tgt gcc ccg gc - #t ggt ttt gcg att cta     
 576                                                                      
Glu Pro Ile Pro Ile His Phe Cys Ala Pro Al - #a Gly Phe Ala Ile Leu       
#           190                                                           
- aag tgt aat aat aag acg ttc gag gga aaa gg - #a cca tgt aaa aat gtc     
 624                                                                      
Lys Cys Asn Asn Lys Thr Phe Glu Gly Lys Gl - #y Pro Cys Lys Asn Val       
#       205                                                               
- agt aca gta caa tgc aca cat gga att agg cc - #a gta gtg tca act caa     
 672                                                                      
Ser Thr Val Gln Cys Thr His Gly Ile Arg Pr - #o Val Val Ser Thr Gln       
#   220                                                                   
- ctg ctg tta aat ggc agt cta gca gaa gaa ga - #g gta ata att aga tct     
 720                                                                      
Leu Leu Leu Asn Gly Ser Leu Ala Glu Glu Gl - #u Val Ile Ile Arg Ser       
225                 2 - #30                 2 - #35                 2 -   
#40                                                                       
- gac aat atc aca gac aat act aaa acc att at - #a gta cag cta aac gaa     
 768                                                                      
Asp Asn Ile Thr Asp Asn Thr Lys Thr Ile Il - #e Val Gln Leu Asn Glu       
#               255                                                       
- tct gta gta att aat tgt aca aga ccc aac aa - #c aat aca aga aaa agt     
 816                                                                      
Ser Val Val Ile Asn Cys Thr Arg Pro Asn As - #n Asn Thr Arg Lys Ser       
#           270                                                           
- ata cat ata gga cca ggg agt gca ttt ttt gc - #a aca gga gaa ata ata     
 864                                                                      
Ile His Ile Gly Pro Gly Ser Ala Phe Phe Al - #a Thr Gly Glu Ile Ile       
#       285                                                               
- gga gat ata aga caa gca cac tgt aac ctt ag - #t aga aca caa tgg aat     
 912                                                                      
Gly Asp Ile Arg Gln Ala His Cys Asn Leu Se - #r Arg Thr Gln Trp Asn       
#   300                                                                   
- aac act tta gga aag ata gtc ata aaa tta ag - #a gaa caa ttt aga aaa     
 960                                                                      
Asn Thr Leu Gly Lys Ile Val Ile Lys Leu Ar - #g Glu Gln Phe Arg Lys       
305                 3 - #10                 3 - #15                 3 -   
#20                                                                       
- caa ttt gga gaa aaa aca ata gtc ttt aat cg - #a tcc tca gga ggg gac     
1008                                                                      
Gln Phe Gly Glu Lys Thr Ile Val Phe Asn Ar - #g Ser Ser Gly Gly Asp       
#               335                                                       
- ccg gaa att gca atg cac agt ttt aat tgt gg - #a ggg gaa ttt ttc tac     
1056                                                                      
Pro Glu Ile Ala Met His Ser Phe Asn Cys Gl - #y Gly Glu Phe Phe Tyr       
#           350                                                           
- tgt aac aca aca gca ctg ttt aat agt acc tg - #g aat gtt act aaa ggg     
1104                                                                      
Cys Asn Thr Thr Ala Leu Phe Asn Ser Thr Tr - #p Asn Val Thr Lys Gly       
#       365                                                               
- ttg aat aac act gaa gga aat agc aca gga ga - #t gaa aat atc ata ctc     
1152                                                                      
Leu Asn Asn Thr Glu Gly Asn Ser Thr Gly As - #p Glu Asn Ile Ile Leu       
#   380                                                                   
- cca tgt aga ata aaa caa att ata aac atg tg - #g cag gaa gta gga aaa     
1200                                                                      
Pro Cys Arg Ile Lys Gln Ile Ile Asn Met Tr - #p Gln Glu Val Gly Lys       
385                 3 - #90                 3 - #95                 4 -   
#00                                                                       
- gca atg tat gcc cct ccc atc agt gga caa at - #t aga tgt tca tca aac     
1248                                                                      
Ala Met Tyr Ala Pro Pro Ile Ser Gly Gln Il - #e Arg Cys Ser Ser Asn       
#               415                                                       
- att aca ggg ctg cta cta aca aga gat ggt gg - #t agt aag aac gag agc     
1296                                                                      
Ile Thr Gly Leu Leu Leu Thr Arg Asp Gly Gl - #y Ser Lys Asn Glu Ser       
#           430                                                           
- atc acc acc gag gtc ttc aga cct gga gga gg - #a gat atg agg gac aat     
1344                                                                      
Ile Thr Thr Glu Val Phe Arg Pro Gly Gly Gl - #y Asp Met Arg Asp Asn       
#       445                                                               
- tgg aga agt gaa tta tat aaa tat aaa gta gt - #a aaa att gaa cca tta     
1392                                                                      
Trp Arg Ser Glu Leu Tyr Lys Tyr Lys Val Va - #l Lys Ile Glu Pro Leu       
#   460                                                                   
- gga gta gcg ccc acc aag gca aag aga aga gt - #g gtg cag aga gaa aaa     
1440                                                                      
Gly Val Ala Pro Thr Lys Ala Lys Arg Arg Va - #l Val Gln Arg Glu Lys       
465                 4 - #70                 4 - #75                 4 -   
#80                                                                       
- aga gca gtg gga aca ata gga gct atg ttc ct - #t ggg ttc ttg gga gca     
1488                                                                      
Arg Ala Val Gly Thr Ile Gly Ala Met Phe Le - #u Gly Phe Leu Gly Ala       
#               495                                                       
#              1512gt cga cct gca                                         
 *  Ser Phe  *  Ser Arg Pro Ala                                           
            500                                                           
- <210> SEQ ID NO 12                                                      
<211> LENGTH: 496                                                         
<212> TYPE: PRT                                                           
<213> ORGANISM: HIV                                                       
- <400> SEQUENCE: 12                                                      
- Leu Glu Val Pro Val Trp Lys Glu Ala Thr Th - #r Thr Leu Phe Cys Ala     
#                15                                                       
- Ser Asp Ala Lys Ala Tyr Asn Thr Glu Lys Hi - #s Asn Val Trp Ala Thr     
#            30                                                           
- His Ala Cys Val Pro Thr Asp Pro Asn Pro Gl - #n Glu Val Val Leu Gly     
#        45                                                               
- Asn Val Thr Glu Asn Phe Asn Met Trp Lys As - #n Asn Met Val Glu Gln     
#    60                                                                   
- Met His Glu Asp Ile Ile Ser Leu Trp Asp Gl - #n Ser Leu Lys Pro Cys     
#80                                                                       
- Val Lys Leu Thr Pro Leu Cys Val Thr Leu As - #n Cys Thr Asp Asp Leu     
#                95                                                       
- Gly Asn Ala Thr Asn Thr Asn Ser Ser Ala Th - #r Thr Asn Ser Ser Ser     
#           110                                                           
- Trp Glu Glu Met Lys Gly Glu Met Lys Arg Cy - #s Ser Phe Asn Ile Thr     
#       125                                                               
- Thr Ser Ile Arg Asp Lys Ile Lys Lys Glu Hi - #s Ala Leu Phe Tyr Arg     
#   140                                                                   
- Leu Asp Val Val Pro Ile Asp Asn Asp Asn Th - #r Thr Tyr Arg Leu Ile     
145                 1 - #50                 1 - #55                 1 -   
#60                                                                       
- Asn Cys Asn Thr Ser Val Ile Thr Gln Ala Cy - #s Pro Lys Val Ser Phe     
#               175                                                       
- Glu Pro Ile Pro Ile His Phe Cys Ala Pro Al - #a Gly Phe Ala Ile Leu     
#           190                                                           
- Lys Cys Asn Asn Lys Thr Phe Glu Gly Lys Gl - #y Pro Cys Lys Asn Val     
#       205                                                               
- Ser Thr Val Gln Cys Thr His Gly Ile Arg Pr - #o Val Val Ser Thr Gln     
#   220                                                                   
- Leu Leu Leu Asn Gly Ser Leu Ala Glu Glu Gl - #u Val Ile Ile Arg Ser     
225                 2 - #30                 2 - #35                 2 -   
#40                                                                       
- Asp Asn Ile Thr Asp Asn Thr Lys Thr Ile Il - #e Val Gln Leu Asn Glu     
#               255                                                       
- Ser Val Val Ile Asn Cys Thr Arg Pro Asn As - #n Asn Thr Arg Lys Ser     
#           270                                                           
- Ile His Ile Gly Pro Gly Ser Ala Phe Phe Al - #a Thr Gly Glu Ile Ile     
#       285                                                               
- Gly Asp Ile Arg Gln Ala His Cys Asn Leu Se - #r Arg Thr Gln Trp Asn     
#   300                                                                   
- Asn Thr Leu Gly Lys Ile Val Ile Lys Leu Ar - #g Glu Gln Phe Arg Lys     
305                 3 - #10                 3 - #15                 3 -   
#20                                                                       
- Gln Phe Gly Glu Lys Thr Ile Val Phe Asn Ar - #g Ser Ser Gly Gly Asp     
#               335                                                       
- Pro Glu Ile Ala Met His Ser Phe Asn Cys Gl - #y Gly Glu Phe Phe Tyr     
#           350                                                           
- Cys Asn Thr Thr Ala Leu Phe Asn Ser Thr Tr - #p Asn Val Thr Lys Gly     
#       365                                                               
- Leu Asn Asn Thr Glu Gly Asn Ser Thr Gly As - #p Glu Asn Ile Ile Leu     
#   380                                                                   
- Pro Cys Arg Ile Lys Gln Ile Ile Asn Met Tr - #p Gln Glu Val Gly Lys     
385                 3 - #90                 3 - #95                 4 -   
#00                                                                       
- Ala Met Tyr Ala Pro Pro Ile Ser Gly Gln Il - #e Arg Cys Ser Ser Asn     
#               415                                                       
- Ile Thr Gly Leu Leu Leu Thr Arg Asp Gly Gl - #y Ser Lys Asn Glu Ser     
#           430                                                           
- Ile Thr Thr Glu Val Phe Arg Pro Gly Gly Gl - #y Asp Met Arg Asp Asn     
#       445                                                               
- Trp Arg Ser Glu Leu Tyr Lys Tyr Lys Val Va - #l Lys Ile Glu Pro Leu     
#   460                                                                   
- Gly Val Ala Pro Thr Lys Ala Lys Arg Arg Va - #l Val Gln Arg Glu Lys     
465                 4 - #70                 4 - #75                 4 -   
#80                                                                       
- Arg Ala Val Gly Thr Ile Gly Ala Met Phe Le - #u Gly Phe Leu Gly Ala     
#               495                                                       
- <210> SEQ ID NO 13                                                      
<211> LENGTH: 2                                                           
<212> TYPE: PRT                                                           
<213> ORGANISM: HIV                                                       
- <400> SEQUENCE: 13                                                      
- Ser Phe                                                                 
 1                                                                        
- <210> SEQ ID NO 14                                                      
<211> LENGTH: 4                                                           
<212> TYPE: PRT                                                           
<213> ORGANISM: HIV                                                       
- <400> SEQUENCE: 14                                                      
- Ser Arg Pro Ala                                                         
 1                                                                        
- <210> SEQ ID NO 15                                                      
<211> LENGTH: 1501                                                        
<212> TYPE: DNA                                                           
<213> ORGANISM: HIV                                                       
<220> FEATURE:                                                            
<221> NAME/KEY: CDS                                                       
<222> LOCATION: (1)...(1500)                                              
- <400> SEQUENCE: 15                                                      
- ctc gag gta cct gtg tgg aaa gaa gca act ac - #c act cta ttt tgt gca     
  48                                                                      
Leu Glu Val Pro Val Trp Lys Glu Ala Thr Th - #r Thr Leu Phe Cys Ala       
#                 15                                                      
- tca gat gct aaa gca tat aat aca gag aaa ca - #t aat gtt tgg gcc aca     
  96                                                                      
Ser Asp Ala Lys Ala Tyr Asn Thr Glu Lys Hi - #s Asn Val Trp Ala Thr       
#             30                                                          
- cac gcc tgt gta ccc aca gat ccc aac cca ca - #a gaa gta gta ttg gga     
 144                                                                      
His Ala Cys Val Pro Thr Asp Pro Asn Pro Gl - #n Glu Val Val Leu Gly       
#         45                                                              
- aat gtg aca gaa aat ttt aac atg tgg aaa aa - #t aac atg gta gaa caa     
 192                                                                      
Asn Val Thr Glu Asn Phe Asn Met Trp Lys As - #n Asn Met Val Glu Gln       
#     60                                                                  
- atg cat gaa gat ata atc agt tta tgg gat ca - #a agt cta aag cca tgt     
 240                                                                      
Met His Glu Asp Ile Ile Ser Leu Trp Asp Gl - #n Ser Leu Lys Pro Cys       
# 80                                                                      
- gta aaa tta acc cca ctc tgt gtt act tta aa - #t tgc act gat gat tta     
 288                                                                      
Val Lys Leu Thr Pro Leu Cys Val Thr Leu As - #n Cys Thr Asp Asp Leu       
#                 95                                                      
- ggg aat gct act aat acc aat agc agt gcc ac - #t acc aat agt agt agt     
 336                                                                      
Gly Asn Ala Thr Asn Thr Asn Ser Ser Ala Th - #r Thr Asn Ser Ser Ser       
#           110                                                           
- tgg gaa gaa atg aag ggg gaa atg aaa agg tg - #c tct ttc aat atc acc     
 384                                                                      
Trp Glu Glu Met Lys Gly Glu Met Lys Arg Cy - #s Ser Phe Asn Ile Thr       
#       125                                                               
- aca agc ata aga gat aag att aag aaa gaa ca - #t gca ctt ttc tat aga     
 432                                                                      
Thr Ser Ile Arg Asp Lys Ile Lys Lys Glu Hi - #s Ala Leu Phe Tyr Arg       
#   140                                                                   
- ctt gat gta gta cca ata gat aat gat aat ac - #c aca tat agg ttg ata     
 480                                                                      
Leu Asp Val Val Pro Ile Asp Asn Asp Asn Th - #r Thr Tyr Arg Leu Ile       
145                 1 - #50                 1 - #55                 1 -   
#60                                                                       
- aat tgt aat acc tca gtc att aca cag gcc tg - #t cca aag gta tca ttt     
 528                                                                      
Asn Cys Asn Thr Ser Val Ile Thr Gln Ala Cy - #s Pro Lys Val Ser Phe       
#               175                                                       
- gag cca att ccc ata cat ttt tgt gcc ccg gc - #t ggt ttt gcg att cta     
 576                                                                      
Glu Pro Ile Pro Ile His Phe Cys Ala Pro Al - #a Gly Phe Ala Ile Leu       
#           190                                                           
- aag tgt aat aat aag acg ttc gag gga aaa gg - #a cca tgt aaa aat gtc     
 624                                                                      
Lys Cys Asn Asn Lys Thr Phe Glu Gly Lys Gl - #y Pro Cys Lys Asn Val       
#       205                                                               
- agt aca gta caa tgc aca cat gga att agg cc - #a gta gtg tca act caa     
 672                                                                      
Ser Thr Val Gln Cys Thr His Gly Ile Arg Pr - #o Val Val Ser Thr Gln       
#   220                                                                   
- ctg ctg tta aat ggc agt cta gca gaa gaa ga - #g gta ata att aga tct     
 720                                                                      
Leu Leu Leu Asn Gly Ser Leu Ala Glu Glu Gl - #u Val Ile Ile Arg Ser       
225                 2 - #30                 2 - #35                 2 -   
#40                                                                       
- ggc aat atc aca gac aat act aaa acc att at - #a gta cag cta aac gaa     
 768                                                                      
Gly Asn Ile Thr Asp Asn Thr Lys Thr Ile Il - #e Val Gln Leu Asn Glu       
#               255                                                       
- tct gta gta att aat tgt aca aga tcc aac aa - #c aat aca aga aaa agt     
 816                                                                      
Ser Val Val Ile Asn Cys Thr Arg Ser Asn As - #n Asn Thr Arg Lys Ser       
#           270                                                           
- ata cat ata gga cca ggg agt gca ttt ttt gc - #a aca gga gaa ata ata     
 864                                                                      
Ile His Ile Gly Pro Gly Ser Ala Phe Phe Al - #a Thr Gly Glu Ile Ile       
#       285                                                               
- gga gat ata aga caa gca cac tgt aac ctt ag - #t aga aca caa tgg aat     
 912                                                                      
Gly Asp Ile Arg Gln Ala His Cys Asn Leu Se - #r Arg Thr Gln Trp Asn       
#   300                                                                   
- aac act tta gga aag ata gtc ata aaa tta ag - #a gaa caa ttt aga aaa     
 960                                                                      
Asn Thr Leu Gly Lys Ile Val Ile Lys Leu Ar - #g Glu Gln Phe Arg Lys       
305                 3 - #10                 3 - #15                 3 -   
#20                                                                       
- caa ttt gga gaa aaa aca ata gtc ttt aat cg - #a tcc tca gga ggg gac     
1008                                                                      
Gln Phe Gly Glu Lys Thr Ile Val Phe Asn Ar - #g Ser Ser Gly Gly Asp       
#               335                                                       
- ccg gaa att gca atg cac agt ttt aat tgt gg - #a ggg gaa ttt ttc tac     
1056                                                                      
Pro Glu Ile Ala Met His Ser Phe Asn Cys Gl - #y Gly Glu Phe Phe Tyr       
#           350                                                           
- tgt aac aca aca gca ctg ttt aat agt acc tg - #g aat gtt act aaa ggg     
1104                                                                      
Cys Asn Thr Thr Ala Leu Phe Asn Ser Thr Tr - #p Asn Val Thr Lys Gly       
#       365                                                               
- ttg aat aac act gaa gga aat agc aca ggg ga - #t gaa aat atc ata ctc     
1152                                                                      
Leu Asn Asn Thr Glu Gly Asn Ser Thr Gly As - #p Glu Asn Ile Ile Leu       
#   380                                                                   
- cca tgt aga ata aaa caa att ata aac atg tg - #g cag gaa gta gga aaa     
1200                                                                      
Pro Cys Arg Ile Lys Gln Ile Ile Asn Met Tr - #p Gln Glu Val Gly Lys       
385                 3 - #90                 3 - #95                 4 -   
#00                                                                       
- gca atg tat gcc cct ccc atc agt gga caa at - #t aga tgt tca tca aat     
1248                                                                      
Ala Met Tyr Ala Pro Pro Ile Ser Gly Gln Il - #e Arg Cys Ser Ser Asn       
#               415                                                       
- att aca ggg ctg cta cta aca aga gat ggt gg - #t agt aag aac gag agc     
1296                                                                      
Ile Thr Gly Leu Leu Leu Thr Arg Asp Gly Gl - #y Ser Lys Asn Glu Ser       
#           430                                                           
- atc acc acc gag gtc ttc aga cct gga gga gg - #a gat atg agg gac aat     
1344                                                                      
Ile Thr Thr Glu Val Phe Arg Pro Gly Gly Gl - #y Asp Met Arg Asp Asn       
#       445                                                               
- tgg aga agt gaa tta tat aaa tat aaa gta gt - #a aaa att gaa cca tta     
1392                                                                      
Trp Arg Ser Glu Leu Tyr Lys Tyr Lys Val Va - #l Lys Ile Glu Pro Leu       
#   460                                                                   
- gga gta gcg ccc acc aag gca aag aga aga gt - #g gtg cag aga gaa aaa     
1440                                                                      
Gly Val Ala Pro Thr Lys Ala Lys Arg Arg Va - #l Val Gln Arg Glu Lys       
465                 4 - #70                 4 - #75                 4 -   
#80                                                                       
- aga gca gtg gga aca ata gga gct atg ttc ct - #t ggg ttc tta gga gca     
1488                                                                      
Arg Ala Val Gly Thr Ile Gly Ala Met Phe Le - #u Gly Phe Leu Gly Ala       
#               495                                                       
#    1501                                                                 
 *  Ser Phe                                                               
- <210> SEQ ID NO 16                                                      
<211> LENGTH: 496                                                         
<212> TYPE: PRT                                                           
<213> ORGANISM: HIV                                                       
- <400> SEQUENCE: 16                                                      
- Leu Glu Val Pro Val Trp Lys Glu Ala Thr Th - #r Thr Leu Phe Cys Ala     
#                15                                                       
- Ser Asp Ala Lys Ala Tyr Asn Thr Glu Lys Hi - #s Asn Val Trp Ala Thr     
#            30                                                           
- His Ala Cys Val Pro Thr Asp Pro Asn Pro Gl - #n Glu Val Val Leu Gly     
#        45                                                               
- Asn Val Thr Glu Asn Phe Asn Met Trp Lys As - #n Asn Met Val Glu Gln     
#    60                                                                   
- Met His Glu Asp Ile Ile Ser Leu Trp Asp Gl - #n Ser Leu Lys Pro Cys     
#80                                                                       
- Val Lys Leu Thr Pro Leu Cys Val Thr Leu As - #n Cys Thr Asp Asp Leu     
#                95                                                       
- Gly Asn Ala Thr Asn Thr Asn Ser Ser Ala Th - #r Thr Asn Ser Ser Ser     
#           110                                                           
- Trp Glu Glu Met Lys Gly Glu Met Lys Arg Cy - #s Ser Phe Asn Ile Thr     
#       125                                                               
- Thr Ser Ile Arg Asp Lys Ile Lys Lys Glu Hi - #s Ala Leu Phe Tyr Arg     
#   140                                                                   
- Leu Asp Val Val Pro Ile Asp Asn Asp Asn Th - #r Thr Tyr Arg Leu Ile     
145                 1 - #50                 1 - #55                 1 -   
#60                                                                       
- Asn Cys Asn Thr Ser Val Ile Thr Gln Ala Cy - #s Pro Lys Val Ser Phe     
#               175                                                       
- Glu Pro Ile Pro Ile His Phe Cys Ala Pro Al - #a Gly Phe Ala Ile Leu     
#           190                                                           
- Lys Cys Asn Asn Lys Thr Phe Glu Gly Lys Gl - #y Pro Cys Lys Asn Val     
#       205                                                               
- Ser Thr Val Gln Cys Thr His Gly Ile Arg Pr - #o Val Val Ser Thr Gln     
#   220                                                                   
- Leu Leu Leu Asn Gly Ser Leu Ala Glu Glu Gl - #u Val Ile Ile Arg Ser     
225                 2 - #30                 2 - #35                 2 -   
#40                                                                       
- Gly Asn Ile Thr Asp Asn Thr Lys Thr Ile Il - #e Val Gln Leu Asn Glu     
#               255                                                       
- Ser Val Val Ile Asn Cys Thr Arg Ser Asn As - #n Asn Thr Arg Lys Ser     
#           270                                                           
- Ile His Ile Gly Pro Gly Ser Ala Phe Phe Al - #a Thr Gly Glu Ile Ile     
#       285                                                               
- Gly Asp Ile Arg Gln Ala His Cys Asn Leu Se - #r Arg Thr Gln Trp Asn     
#   300                                                                   
- Asn Thr Leu Gly Lys Ile Val Ile Lys Leu Ar - #g Glu Gln Phe Arg Lys     
305                 3 - #10                 3 - #15                 3 -   
#20                                                                       
- Gln Phe Gly Glu Lys Thr Ile Val Phe Asn Ar - #g Ser Ser Gly Gly Asp     
#               335                                                       
- Pro Glu Ile Ala Met His Ser Phe Asn Cys Gl - #y Gly Glu Phe Phe Tyr     
#           350                                                           
- Cys Asn Thr Thr Ala Leu Phe Asn Ser Thr Tr - #p Asn Val Thr Lys Gly     
#       365                                                               
- Leu Asn Asn Thr Glu Gly Asn Ser Thr Gly As - #p Glu Asn Ile Ile Leu     
#   380                                                                   
- Pro Cys Arg Ile Lys Gln Ile Ile Asn Met Tr - #p Gln Glu Val Gly Lys     
385                 3 - #90                 3 - #95                 4 -   
#00                                                                       
- Ala Met Tyr Ala Pro Pro Ile Ser Gly Gln Il - #e Arg Cys Ser Ser Asn     
#               415                                                       
- Ile Thr Gly Leu Leu Leu Thr Arg Asp Gly Gl - #y Ser Lys Asn Glu Ser     
#           430                                                           
- Ile Thr Thr Glu Val Phe Arg Pro Gly Gly Gl - #y Asp Met Arg Asp Asn     
#       445                                                               
- Trp Arg Ser Glu Leu Tyr Lys Tyr Lys Val Va - #l Lys Ile Glu Pro Leu     
#   460                                                                   
- Gly Val Ala Pro Thr Lys Ala Lys Arg Arg Va - #l Val Gln Arg Glu Lys     
465                 4 - #70                 4 - #75                 4 -   
#80                                                                       
- Arg Ala Val Gly Thr Ile Gly Ala Met Phe Le - #u Gly Phe Leu Gly Ala     
#               495                                                       
- <210> SEQ ID NO 17                                                      
<211> LENGTH: 2                                                           
<212> TYPE: PRT                                                           
<213> ORGANISM: HIV                                                       
- <400> SEQUENCE: 17                                                      
- Ser Phe                                                                 
 1                                                                        
- <210> SEQ ID NO 18                                                      
<211> LENGTH: 1514                                                        
<212> TYPE: DNA                                                           
<213> ORGANISM: HIV                                                       
<220> FEATURE:                                                            
<221> NAME/KEY: CDS                                                       
<222> LOCATION: (3)...(1514)                                              
- <400> SEQUENCE: 18                                                      
- gg gaa ttc gga tcc ggg gta cct gtg tgg aag - # gaa gca acc acc act      
  47                                                                      
#Lys Glu Ala Thr Thr Thral Pro Val Trp                                    
#  15                                                                     
- cta ttc tgt gca tca gat gct aga gca tat ga - #c aca gag gta cat aat     
  95                                                                      
Leu Phe Cys Ala Ser Asp Ala Arg Ala Tyr As - #p Thr Glu Val His Asn       
#                 30                                                      
- gtt tgg gcc aca cat gcc tgt gta ccc aca ga - #c cct agt cca caa gaa     
 143                                                                      
Val Trp Ala Thr His Ala Cys Val Pro Thr As - #p Pro Ser Pro Gln Glu       
#             45                                                          
- gta gtt ttg gaa aat gtg aca gaa aat ttt aa - #c atg tgg aaa aat aac     
 191                                                                      
Val Val Leu Glu Asn Val Thr Glu Asn Phe As - #n Met Trp Lys Asn Asn       
#         60                                                              
- atg gta gaa caa atg cat gag gat ata att ag - #t tta tgg gat caa agc     
 239                                                                      
Met Val Glu Gln Met His Glu Asp Ile Ile Se - #r Leu Trp Asp Gln Ser       
#     75                                                                  
- tta aag cca tgt gta aaa tta acc cca ctc tg - #t gtt act tta aat tgc     
 287                                                                      
Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cy - #s Val Thr Leu Asn Cys       
# 95                                                                      
- agt gat tat agg aat gct act gat tat aag aa - #t gct act gat acc act     
 335                                                                      
Ser Asp Tyr Arg Asn Ala Thr Asp Tyr Lys As - #n Ala Thr Asp Thr Thr       
#               110                                                       
- agt agt aac gag gga aag atg gag aga gga ga - #a ata aaa aac tgc tct     
 383                                                                      
Ser Ser Asn Glu Gly Lys Met Glu Arg Gly Gl - #u Ile Lys Asn Cys Ser       
#           125                                                           
- ttc aat att acc aca agc ata aaa aat aag at - #g cag aaa gaa tat gca     
 431                                                                      
Phe Asn Ile Thr Thr Ser Ile Lys Asn Lys Me - #t Gln Lys Glu Tyr Ala       
#       140                                                               
- ctt ttc tat aaa ctt gat ata gta cca ata ga - #t aat aca agc tat aca     
 479                                                                      
Leu Phe Tyr Lys Leu Asp Ile Val Pro Ile As - #p Asn Thr Ser Tyr Thr       
#   155                                                                   
- ttg ata agt tgt aac acc tca gtc att aca ca - #g gcc tgt cca aag gta     
 527                                                                      
Leu Ile Ser Cys Asn Thr Ser Val Ile Thr Gl - #n Ala Cys Pro Lys Val       
160                 1 - #65                 1 - #70                 1 -   
#75                                                                       
- tcc ttt gaa cca act ccc ata cat tat tgt gc - #t ccg gct ggt ttt gcg     
 575                                                                      
Ser Phe Glu Pro Thr Pro Ile His Tyr Cys Al - #a Pro Ala Gly Phe Ala       
#               190                                                       
- att cta aag tgt aat gat aag aag ttc agt gg - #a aaa gga gaa tgt aaa     
 623                                                                      
Ile Leu Lys Cys Asn Asp Lys Lys Phe Ser Gl - #y Lys Gly Glu Cys Lys       
#           205                                                           
- aat gtc agc aca gta caa tgt aca cat gga at - #t agg cca gta gta tca     
 671                                                                      
Asn Val Ser Thr Val Gln Cys Thr His Gly Il - #e Arg Pro Val Val Ser       
#       220                                                               
- act caa ctg ctg tta aat ggc agt cta gca ga - #a gaa gag gtg gta att     
 719                                                                      
Thr Gln Leu Leu Leu Asn Gly Ser Leu Ala Gl - #u Glu Glu Val Val Ile       
#   235                                                                   
- aga tct gac aat ttc ata gac aat act aaa ac - #c ata ata gta cag ctg     
 767                                                                      
Arg Ser Asp Asn Phe Ile Asp Asn Thr Lys Th - #r Ile Ile Val Gln Leu       
240                 2 - #45                 2 - #50                 2 -   
#55                                                                       
- aaa gaa tct gta gaa att aat tgt ata aga cc - #c aac aat aat aca aga     
 815                                                                      
Lys Glu Ser Val Glu Ile Asn Cys Ile Arg Pr - #o Asn Asn Asn Thr Arg       
#               270                                                       
- aaa ggt ata cat ata gga cca ggg aga gca tg - #g tat gca aca gga gaa     
 863                                                                      
Lys Gly Ile His Ile Gly Pro Gly Arg Ala Tr - #p Tyr Ala Thr Gly Glu       
#           285                                                           
- ata gta gga gat ata aga aag gca tat tgt aa - #c att agt aga aca aaa     
 911                                                                      
Ile Val Gly Asp Ile Arg Lys Ala Tyr Cys As - #n Ile Ser Arg Thr Lys       
#       300                                                               
- tgg aat aac act tta ata cag ata gct aac aa - #a tta aaa gaa aaa tat     
 959                                                                      
Trp Asn Asn Thr Leu Ile Gln Ile Ala Asn Ly - #s Leu Lys Glu Lys Tyr       
#   315                                                                   
- aat aca aca ata agc ttt aat cga tcc tca gg - #a ggg gac cca gaa att     
1007                                                                      
Asn Thr Thr Ile Ser Phe Asn Arg Ser Ser Gl - #y Gly Asp Pro Glu Ile       
320                 3 - #25                 3 - #30                 3 -   
#35                                                                       
- gta acg cat agt ttt aat tgt gga ggg gag tt - #t ttc tac tgt gat tca     
1055                                                                      
Val Thr His Ser Phe Asn Cys Gly Gly Glu Ph - #e Phe Tyr Cys Asp Ser       
#               350                                                       
- aca caa ctg ttt aat agt act tgg aat tta aa - #t ggt act tgg aat ttt     
1103                                                                      
Thr Gln Leu Phe Asn Ser Thr Trp Asn Leu As - #n Gly Thr Trp Asn Phe       
#           365                                                           
- act gca ggg tca aat gaa act gaa ggc aat at - #c aca ctc cca tgc aga     
1151                                                                      
Thr Ala Gly Ser Asn Glu Thr Glu Gly Asn Il - #e Thr Leu Pro Cys Arg       
#       380                                                               
- ata aaa caa att ata aac agg tgg cag gaa gt - #a ggg aaa gca atg tat     
1199                                                                      
Ile Lys Gln Ile Ile Asn Arg Trp Gln Glu Va - #l Gly Lys Ala Met Tyr       
#   395                                                                   
- gcc cct ccc atc agt gga caa ata aaa tgc tc - #a tca aac att aca ggg     
1247                                                                      
Ala Pro Pro Ile Ser Gly Gln Ile Lys Cys Se - #r Ser Asn Ile Thr Gly       
400                 4 - #05                 4 - #10                 4 -   
#15                                                                       
- atg ata tta aca agg gat ggt ggt aac gag aa - #c aat aat gag agc agt     
1295                                                                      
Met Ile Leu Thr Arg Asp Gly Gly Asn Glu As - #n Asn Asn Glu Ser Ser       
#               430                                                       
- act act gag acc ttc aga ccg gga gga gga ga - #t atg agg aac aat tgg     
1343                                                                      
Thr Thr Glu Thr Phe Arg Pro Gly Gly Gly As - #p Met Arg Asn Asn Trp       
#           445                                                           
- aga agt gaa tta tat aaa tat aaa gta gta aa - #a att gaa cca tta gga     
1391                                                                      
Arg Ser Glu Leu Tyr Lys Tyr Lys Val Val Ly - #s Ile Glu Pro Leu Gly       
#       460                                                               
- gta gca ccc acc aag gca aag aga aga gtg gt - #g cag aga gaa aaa aga     
1439                                                                      
Val Ala Pro Thr Lys Ala Lys Arg Arg Val Va - #l Gln Arg Glu Lys Arg       
#   475                                                                   
- gca gtg gga gcg cta gga gct atg ttc ctt gg - #g ttc tta gga gca taa     
1487                                                                      
Ala Val Gly Ala Leu Gly Ala Met Phe Leu Gl - #y Phe Leu Gly Ala           
480                 4 - #85                 4 - #90                 4 -   
#95                                                                       
#           1514   ac tct aga gga tcc                                     
Ser Phe  *  Thr Asp Ser Arg Gly Ser                                       
                500                                                       
- <210> SEQ ID NO 19                                                      
<211> LENGTH: 494                                                         
<212> TYPE: PRT                                                           
<213> ORGANISM: HIV                                                       
- <400> SEQUENCE: 19                                                      
- Glu Phe Gly Ser Gly Val Pro Val Trp Lys Gl - #u Ala Thr Thr Thr Leu     
#                15                                                       
- Phe Cys Ala Ser Asp Ala Arg Ala Tyr Asp Th - #r Glu Val His Asn Val     
#            30                                                           
- Trp Ala Thr His Ala Cys Val Pro Thr Asp Pr - #o Ser Pro Gln Glu Val     
#        45                                                               
- Val Leu Glu Asn Val Thr Glu Asn Phe Asn Me - #t Trp Lys Asn Asn Met     
#    60                                                                   
- Val Glu Gln Met His Glu Asp Ile Ile Ser Le - #u Trp Asp Gln Ser Leu     
#80                                                                       
- Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Va - #l Thr Leu Asn Cys Ser     
#                95                                                       
- Asp Tyr Arg Asn Ala Thr Asp Tyr Lys Asn Al - #a Thr Asp Thr Thr Ser     
#           110                                                           
- Ser Asn Glu Gly Lys Met Glu Arg Gly Glu Il - #e Lys Asn Cys Ser Phe     
#       125                                                               
- Asn Ile Thr Thr Ser Ile Lys Asn Lys Met Gl - #n Lys Glu Tyr Ala Leu     
#   140                                                                   
- Phe Tyr Lys Leu Asp Ile Val Pro Ile Asp As - #n Thr Ser Tyr Thr Leu     
145                 1 - #50                 1 - #55                 1 -   
#60                                                                       
- Ile Ser Cys Asn Thr Ser Val Ile Thr Gln Al - #a Cys Pro Lys Val Ser     
#               175                                                       
- Phe Glu Pro Thr Pro Ile His Tyr Cys Ala Pr - #o Ala Gly Phe Ala Ile     
#           190                                                           
- Leu Lys Cys Asn Asp Lys Lys Phe Ser Gly Ly - #s Gly Glu Cys Lys Asn     
#       205                                                               
- Val Ser Thr Val Gln Cys Thr His Gly Ile Ar - #g Pro Val Val Ser Thr     
#   220                                                                   
- Gln Leu Leu Leu Asn Gly Ser Leu Ala Glu Gl - #u Glu Val Val Ile Arg     
225                 2 - #30                 2 - #35                 2 -   
#40                                                                       
- Ser Asp Asn Phe Ile Asp Asn Thr Lys Thr Il - #e Ile Val Gln Leu Lys     
#               255                                                       
- Glu Ser Val Glu Ile Asn Cys Ile Arg Pro As - #n Asn Asn Thr Arg Lys     
#           270                                                           
- Gly Ile His Ile Gly Pro Gly Arg Ala Trp Ty - #r Ala Thr Gly Glu Ile     
#       285                                                               
- Val Gly Asp Ile Arg Lys Ala Tyr Cys Asn Il - #e Ser Arg Thr Lys Trp     
#   300                                                                   
- Asn Asn Thr Leu Ile Gln Ile Ala Asn Lys Le - #u Lys Glu Lys Tyr Asn     
305                 3 - #10                 3 - #15                 3 -   
#20                                                                       
- Thr Thr Ile Ser Phe Asn Arg Ser Ser Gly Gl - #y Asp Pro Glu Ile Val     
#               335                                                       
- Thr His Ser Phe Asn Cys Gly Gly Glu Phe Ph - #e Tyr Cys Asp Ser Thr     
#           350                                                           
- Gln Leu Phe Asn Ser Thr Trp Asn Leu Asn Gl - #y Thr Trp Asn Phe Thr     
#       365                                                               
- Ala Gly Ser Asn Glu Thr Glu Gly Asn Ile Th - #r Leu Pro Cys Arg Ile     
#   380                                                                   
- Lys Gln Ile Ile Asn Arg Trp Gln Glu Val Gl - #y Lys Ala Met Tyr Ala     
385                 3 - #90                 3 - #95                 4 -   
#00                                                                       
- Pro Pro Ile Ser Gly Gln Ile Lys Cys Ser Se - #r Asn Ile Thr Gly Met     
#               415                                                       
- Ile Leu Thr Arg Asp Gly Gly Asn Glu Asn As - #n Asn Glu Ser Ser Thr     
#           430                                                           
- Thr Glu Thr Phe Arg Pro Gly Gly Gly Asp Me - #t Arg Asn Asn Trp Arg     
#       445                                                               
- Ser Glu Leu Tyr Lys Tyr Lys Val Val Lys Il - #e Glu Pro Leu Gly Val     
#   460                                                                   
- Ala Pro Thr Lys Ala Lys Arg Arg Val Val Gl - #n Arg Glu Lys Arg Ala     
465                 4 - #70                 4 - #75                 4 -   
#80                                                                       
- Val Gly Ala Leu Gly Ala Met Phe Leu Gly Ph - #e Leu Gly Ala             
#               490                                                       
- <210> SEQ ID NO 20                                                      
<211> LENGTH: 2                                                           
<212> TYPE: PRT                                                           
<213> ORGANISM: HIV                                                       
- <400> SEQUENCE: 20                                                      
- Ser Phe                                                                 
 1                                                                        
- <210> SEQ ID NO 21                                                      
<211> LENGTH: 6                                                           
<212> TYPE: PRT                                                           
<213> ORGANISM: HIV                                                       
- <400> SEQUENCE: 21                                                      
- Thr Asp Ser Arg Gly Ser                                                 
 1               5                                                        
- <210> SEQ ID NO 22                                                      
<211> LENGTH: 1408                                                        
<212> TYPE: DNA                                                           
<213> ORGANISM: HIV                                                       
<220> FEATURE:                                                            
<221> NAME/KEY: CDS                                                       
<222> LOCATION: (2)...(1408)                                              
- <400> SEQUENCE: 22                                                      
#cta ttc tgt gca tca gat     49 acc acc act                               
  Val Pro Val Trp Lys Glu Ala Thr Thr T - #hr Leu Phe Cys Ala Ser Asp     
# 15                                                                      
- gct aga gca tat gac aca gag gta cat aat gt - #t tgg gcc aca cat gcc     
  97                                                                      
Ala Arg Ala Tyr Asp Thr Glu Val His Asn Va - #l Trp Ala Thr His Ala       
#             30                                                          
- tgt gta ccc aca gac cct agt cca caa gaa gt - #a ttt ttg gga aat gtg     
 145                                                                      
Cys Val Pro Thr Asp Pro Ser Pro Gln Glu Va - #l Phe Leu Gly Asn Val       
#         45                                                              
- aca gaa aat ttt aat atg tgg aaa aat aac at - #g gta gaa caa atg tat     
 193                                                                      
Thr Glu Asn Phe Asn Met Trp Lys Asn Asn Me - #t Val Glu Gln Met Tyr       
#     60                                                                  
- gag gat ata att agt tta tgg gat caa agc tt - #a aag cca tgt gta aaa     
 241                                                                      
Glu Asp Ile Ile Ser Leu Trp Asp Gln Ser Le - #u Lys Pro Cys Val Lys       
# 80                                                                      
- tta acc cca ctc tgt gtt act tta aat tgc ag - #t gat tat agg aat gct     
 289                                                                      
Leu Thr Pro Leu Cys Val Thr Leu Asn Cys Se - #r Asp Tyr Arg Asn Ala       
#                 95                                                      
- act gat tat aag aat gct act gat acc act ag - #t agt aac gag gga aag     
 337                                                                      
Thr Asp Tyr Lys Asn Ala Thr Asp Thr Thr Se - #r Ser Asn Glu Gly Lys       
#           110                                                           
- atg gag aga gga gaa ata aaa aac tgc tct tt - #c aat atc acc aca agc     
 385                                                                      
Met Glu Arg Gly Glu Ile Lys Asn Cys Ser Ph - #e Asn Ile Thr Thr Ser       
#       125                                                               
- ata aaa aat aag atg cag aaa gaa tat gca ct - #t ttc tat aaa ctt aat     
 433                                                                      
Ile Lys Asn Lys Met Gln Lys Glu Tyr Ala Le - #u Phe Tyr Lys Leu Asn       
#   140                                                                   
- ata gta cca ata gat aat aca agc tat aca tt - #g ata agt tgt aac acc     
 481                                                                      
Ile Val Pro Ile Asp Asn Thr Ser Tyr Thr Le - #u Ile Ser Cys Asn Thr       
145                 1 - #50                 1 - #55                 1 -   
#60                                                                       
- tca gtc att aca cag gcc tgt cca aag gta tc - #c ttt gaa cca att ccc     
 529                                                                      
Ser Val Ile Thr Gln Ala Cys Pro Lys Val Se - #r Phe Glu Pro Ile Pro       
#               175                                                       
- ata cat tat tgt gct ccg gct ggt ttt gcg at - #t cta aag tgt aat gat     
 577                                                                      
Ile His Tyr Cys Ala Pro Ala Gly Phe Ala Il - #e Leu Lys Cys Asn Asp       
#           190                                                           
- aag aag ttc agt gga aaa gga gaa tgt aaa aa - #t gtc agc aca gta caa     
 625                                                                      
Lys Lys Phe Ser Gly Lys Gly Glu Cys Lys As - #n Val Ser Thr Val Gln       
#       205                                                               
- tgt aca cat gga att agg cca gta gta tca ac - #t caa ctg ctg tta aat     
 673                                                                      
Cys Thr His Gly Ile Arg Pro Val Val Ser Th - #r Gln Leu Leu Leu Asn       
#   220                                                                   
- ggc agt cta gca gaa gaa gag gtg gta att ag - #a tct gac aat ttc aca     
 721                                                                      
Gly Ser Leu Ala Glu Glu Glu Val Val Ile Ar - #g Ser Asp Asn Phe Thr       
225                 2 - #30                 2 - #35                 2 -   
#40                                                                       
- gac aat act aaa acc ata ata gta cag ctg aa - #a gaa tct gta gaa att     
 769                                                                      
Asp Asn Thr Lys Thr Ile Ile Val Gln Leu Ly - #s Glu Ser Val Glu Ile       
#               255                                                       
- aat tgt ata aga ccc aac aat aat aca aga aa - #a ggt ata cat ata gga     
 817                                                                      
Asn Cys Ile Arg Pro Asn Asn Asn Thr Arg Ly - #s Gly Ile His Ile Gly       
#           270                                                           
- cca ggg aga gca tgg tat gca aca gga gaa at - #a gta gga gat ata aga     
 865                                                                      
Pro Gly Arg Ala Trp Tyr Ala Thr Gly Glu Il - #e Val Gly Asp Ile Arg       
#       285                                                               
- cag gca tat tgt aac att agt aga aca aaa tg - #g aat aac act tta ata     
 913                                                                      
Gln Ala Tyr Cys Asn Ile Ser Arg Thr Lys Tr - #p Asn Asn Thr Leu Ile       
#   300                                                                   
- cag ata gct aac aaa tta aaa gaa aaa tat aa - #t aca aca ata agc ttt     
 961                                                                      
Gln Ile Ala Asn Lys Leu Lys Glu Lys Tyr As - #n Thr Thr Ile Ser Phe       
305                 3 - #10                 3 - #15                 3 -   
#20                                                                       
- aat cga tcc tca gga ggg gac cca gaa att gt - #a acc cat agt ttt aat     
1009                                                                      
Asn Arg Ser Ser Gly Gly Asp Pro Glu Ile Va - #l Thr His Ser Phe Asn       
#               335                                                       
- tgt gga ggg gaa ttt ttc tac tgt aat tca ac - #a caa ctg ttt aat agt     
1057                                                                      
Cys Gly Gly Glu Phe Phe Tyr Cys Asn Ser Th - #r Gln Leu Phe Asn Ser       
#           350                                                           
- act tgg aat tta aat ggt act tgg aat ttt ac - #t gca ggg tca aat gaa     
1105                                                                      
Thr Trp Asn Leu Asn Gly Thr Trp Asn Phe Th - #r Ala Gly Ser Asn Glu       
#       365                                                               
- act gaa ggc aat atc aca ctc cca tgc aga at - #a aaa caa att ata aac     
1153                                                                      
Thr Glu Gly Asn Ile Thr Leu Pro Cys Arg Il - #e Lys Gln Ile Ile Asn       
#   380                                                                   
- agg tgg cag gaa gta gga aaa gca atg tat gc - #c cct ccc atc agt gga     
1201                                                                      
Arg Trp Gln Glu Val Gly Lys Ala Met Tyr Al - #a Pro Pro Ile Ser Gly       
385                 3 - #90                 3 - #95                 4 -   
#00                                                                       
- caa ata aga tgc tca tca aac att aca ggg at - #g ata tta aca agg gat     
1249                                                                      
Gln Ile Arg Cys Ser Ser Asn Ile Thr Gly Me - #t Ile Leu Thr Arg Asp       
#               415                                                       
- ggt ggt aac gag aac aat aat gag agc agt ac - #t act gag acc ttc aga     
1297                                                                      
Gly Gly Asn Glu Asn Asn Asn Glu Ser Ser Th - #r Thr Glu Thr Phe Arg       
#           430                                                           
- ccg gga gga gga gat atg agg aac aat tgg ag - #a agt gaa tta tat aaa     
1345                                                                      
Pro Gly Gly Gly Asp Met Arg Asn Asn Trp Ar - #g Ser Glu Leu Tyr Lys       
#       445                                                               
- tat aaa gta gta aaa att gag cca tta gga gt - #a gca ccc acc gac tct     
1393                                                                      
Tyr Lys Val Val Lys Ile Glu Pro Leu Gly Va - #l Ala Pro Thr Asp Ser       
#   460                                                                   
#  1408            ga                                                     
Arg Gly Ser Ser Arg                                                       
465                                                                       
- <210> SEQ ID NO 23                                                      
<211> LENGTH: 469                                                         
<212> TYPE: PRT                                                           
<213> ORGANISM: HIV                                                       
- <400> SEQUENCE: 23                                                      
- Val Pro Val Trp Lys Glu Ala Thr Thr Thr Le - #u Phe Cys Ala Ser Asp     
#                15                                                       
- Ala Arg Ala Tyr Asp Thr Glu Val His Asn Va - #l Trp Ala Thr His Ala     
#            30                                                           
- Cys Val Pro Thr Asp Pro Ser Pro Gln Glu Va - #l Phe Leu Gly Asn Val     
#        45                                                               
- Thr Glu Asn Phe Asn Met Trp Lys Asn Asn Me - #t Val Glu Gln Met Tyr     
#    60                                                                   
- Glu Asp Ile Ile Ser Leu Trp Asp Gln Ser Le - #u Lys Pro Cys Val Lys     
#80                                                                       
- Leu Thr Pro Leu Cys Val Thr Leu Asn Cys Se - #r Asp Tyr Arg Asn Ala     
#                95                                                       
- Thr Asp Tyr Lys Asn Ala Thr Asp Thr Thr Se - #r Ser Asn Glu Gly Lys     
#           110                                                           
- Met Glu Arg Gly Glu Ile Lys Asn Cys Ser Ph - #e Asn Ile Thr Thr Ser     
#       125                                                               
- Ile Lys Asn Lys Met Gln Lys Glu Tyr Ala Le - #u Phe Tyr Lys Leu Asn     
#   140                                                                   
- Ile Val Pro Ile Asp Asn Thr Ser Tyr Thr Le - #u Ile Ser Cys Asn Thr     
145                 1 - #50                 1 - #55                 1 -   
#60                                                                       
- Ser Val Ile Thr Gln Ala Cys Pro Lys Val Se - #r Phe Glu Pro Ile Pro     
#               175                                                       
- Ile His Tyr Cys Ala Pro Ala Gly Phe Ala Il - #e Leu Lys Cys Asn Asp     
#           190                                                           
- Lys Lys Phe Ser Gly Lys Gly Glu Cys Lys As - #n Val Ser Thr Val Gln     
#       205                                                               
- Cys Thr His Gly Ile Arg Pro Val Val Ser Th - #r Gln Leu Leu Leu Asn     
#   220                                                                   
- Gly Ser Leu Ala Glu Glu Glu Val Val Ile Ar - #g Ser Asp Asn Phe Thr     
225                 2 - #30                 2 - #35                 2 -   
#40                                                                       
- Asp Asn Thr Lys Thr Ile Ile Val Gln Leu Ly - #s Glu Ser Val Glu Ile     
#               255                                                       
- Asn Cys Ile Arg Pro Asn Asn Asn Thr Arg Ly - #s Gly Ile His Ile Gly     
#           270                                                           
- Pro Gly Arg Ala Trp Tyr Ala Thr Gly Glu Il - #e Val Gly Asp Ile Arg     
#       285                                                               
- Gln Ala Tyr Cys Asn Ile Ser Arg Thr Lys Tr - #p Asn Asn Thr Leu Ile     
#   300                                                                   
- Gln Ile Ala Asn Lys Leu Lys Glu Lys Tyr As - #n Thr Thr Ile Ser Phe     
305                 3 - #10                 3 - #15                 3 -   
#20                                                                       
- Asn Arg Ser Ser Gly Gly Asp Pro Glu Ile Va - #l Thr His Ser Phe Asn     
#               335                                                       
- Cys Gly Gly Glu Phe Phe Tyr Cys Asn Ser Th - #r Gln Leu Phe Asn Ser     
#           350                                                           
- Thr Trp Asn Leu Asn Gly Thr Trp Asn Phe Th - #r Ala Gly Ser Asn Glu     
#       365                                                               
- Thr Glu Gly Asn Ile Thr Leu Pro Cys Arg Il - #e Lys Gln Ile Ile Asn     
#   380                                                                   
- Arg Trp Gln Glu Val Gly Lys Ala Met Tyr Al - #a Pro Pro Ile Ser Gly     
385                 3 - #90                 3 - #95                 4 -   
#00                                                                       
- Gln Ile Arg Cys Ser Ser Asn Ile Thr Gly Me - #t Ile Leu Thr Arg Asp     
#               415                                                       
- Gly Gly Asn Glu Asn Asn Asn Glu Ser Ser Th - #r Thr Glu Thr Phe Arg     
#           430                                                           
- Pro Gly Gly Gly Asp Met Arg Asn Asn Trp Ar - #g Ser Glu Leu Tyr Lys     
#       445                                                               
- Tyr Lys Val Val Lys Ile Glu Pro Leu Gly Va - #l Ala Pro Thr Asp Ser     
#   460                                                                   
- Arg Gly Ser Ser Arg                                                     
465                                                                       
- <210> SEQ ID NO 24                                                      
<211> LENGTH: 1499                                                        
<212> TYPE: DNA                                                           
<213> ORGANISM: HIV                                                       
<220> FEATURE:                                                            
<221> NAME/KEY: CDS                                                       
<222> LOCATION: (1)...(1497)                                              
- <400> SEQUENCE: 24                                                      
- gag gta cct gtg tgg aaa gaa gca acc act ac - #t cta ttt tgt gca tca     
  48                                                                      
Glu Val Pro Val Trp Lys Glu Ala Thr Thr Th - #r Leu Phe Cys Ala Ser       
#                 15                                                      
- gat gct aaa gca tat gac aca ggg gtg cat aa - #t gtt tgg gcc aca cat     
  96                                                                      
Asp Ala Lys Ala Tyr Asp Thr Gly Val His As - #n Val Trp Ala Thr His       
#             30                                                          
- gcc tgt gta ccc aca gac ccc aac cca caa ga - #a ata gaa ttg gta aat     
 144                                                                      
Ala Cys Val Pro Thr Asp Pro Asn Pro Gln Gl - #u Ile Glu Leu Val Asn       
#         45                                                              
- gtg aca gaa gat ttt aac atg tgg aaa aat aa - #a atg gta gac cag atg     
 192                                                                      
Val Thr Glu Asp Phe Asn Met Trp Lys Asn Ly - #s Met Val Asp Gln Met       
#     60                                                                  
- cat gag gat ata atc agt tta tgg gat gaa ag - #c cta aag cca tgt gta     
 240                                                                      
His Glu Asp Ile Ile Ser Leu Trp Asp Glu Se - #r Leu Lys Pro Cys Val       
# 80                                                                      
- aag tta acc cca ctt tgt gtt act cta aac tg - #c agt gat gtg aac aat     
 288                                                                      
Lys Leu Thr Pro Leu Cys Val Thr Leu Asn Cy - #s Ser Asp Val Asn Asn       
#                 95                                                      
- tcc aca aat cct aat gat act aat act aat tc - #c act aat act act tcc     
 336                                                                      
Ser Thr Asn Pro Asn Asp Thr Asn Thr Asn Se - #r Thr Asn Thr Thr Ser       
#           110                                                           
- tct act cct acg gcc act act agt agc gag ga - #a aag atg gag aag gga     
 384                                                                      
Ser Thr Pro Thr Ala Thr Thr Ser Ser Glu Gl - #u Lys Met Glu Lys Gly       
#       125                                                               
- gaa ata aaa aac tgc tct ttc aat atc acc ac - #a cac atg aaa gat aag     
 432                                                                      
Glu Ile Lys Asn Cys Ser Phe Asn Ile Thr Th - #r His Met Lys Asp Lys       
#   140                                                                   
- gca cag aaa gaa tat gca ctt ttt tat aaa ct - #t gat ata gta cca ata     
 480                                                                      
Ala Gln Lys Glu Tyr Ala Leu Phe Tyr Lys Le - #u Asp Ile Val Pro Ile       
145                 1 - #50                 1 - #55                 1 -   
#60                                                                       
- gat gat aat aat gcc agc tat agg ttg ata ag - #t tgt aat acc tca gac     
 528                                                                      
Asp Asp Asn Asn Ala Ser Tyr Arg Leu Ile Se - #r Cys Asn Thr Ser Asp       
#               175                                                       
- att aca cag gcc tgt cca aag gtg acc ttt ga - #g cca att ccc ata cat     
 576                                                                      
Ile Thr Gln Ala Cys Pro Lys Val Thr Phe Gl - #u Pro Ile Pro Ile His       
#           190                                                           
- tat tgt gcc ccg gct ggt ttt gcg att cta aa - #g tgt aaa gat aag aag     
 624                                                                      
Tyr Cys Ala Pro Ala Gly Phe Ala Ile Leu Ly - #s Cys Lys Asp Lys Lys       
#       205                                                               
- ttc aat gga aca gga cca tgt tca aag gtc ag - #c aca gta caa tgt aca     
 672                                                                      
Phe Asn Gly Thr Gly Pro Cys Ser Lys Val Se - #r Thr Val Gln Cys Thr       
#   220                                                                   
- cat gga att agg cca gta gta tca act caa ct - #g ttg tta aat ggc agt     
 720                                                                      
His Gly Ile Arg Pro Val Val Ser Thr Gln Le - #u Leu Leu Asn Gly Ser       
225                 2 - #30                 2 - #35                 2 -   
#40                                                                       
- ctt gca gaa gaa gaa gta gta att aga tct gt - #c aat ttc aca gac aat     
 768                                                                      
Leu Ala Glu Glu Glu Val Val Ile Arg Ser Va - #l Asn Phe Thr Asp Asn       
#               255                                                       
- gct aaa atc ata ata gta cag ctg aaa gaa cc - #t gta gca att aat tgt     
 816                                                                      
Ala Lys Ile Ile Ile Val Gln Leu Lys Glu Pr - #o Val Ala Ile Asn Cys       
#           270                                                           
- aca aga ccc aac aac aat aca aga aaa ggt at - #a cat cta gga cca ggg     
 864                                                                      
Thr Arg Pro Asn Asn Asn Thr Arg Lys Gly Il - #e His Leu Gly Pro Gly       
#       285                                                               
- agc aca ttt tat aca aca gga gaa ata ata gg - #a gac ata aga aaa gca     
 912                                                                      
Ser Thr Phe Tyr Thr Thr Gly Glu Ile Ile Gl - #y Asp Ile Arg Lys Ala       
#   300                                                                   
- tat tgc aag att agt aaa gaa aaa tgg aat aa - #c act tta aga cag gta     
 960                                                                      
Tyr Cys Lys Ile Ser Lys Glu Lys Trp Asn As - #n Thr Leu Arg Gln Val       
305                 3 - #10                 3 - #15                 3 -   
#20                                                                       
- gtt aaa aaa tta aga gaa caa ttt ggg aat aa - #a aca ata att ttt aat     
1008                                                                      
Val Lys Lys Leu Arg Glu Gln Phe Gly Asn Ly - #s Thr Ile Ile Phe Asn       
#               335                                                       
- cga tcc tca gga ggg gac cca gaa att gta at - #g cac agt ttt aac tgt     
1056                                                                      
Arg Ser Ser Gly Gly Asp Pro Glu Ile Val Me - #t His Ser Phe Asn Cys       
#           350                                                           
- gga ggg gag ttt ttc tac tgt aat aca aca ca - #a ctg ttt aat agt act     
1104                                                                      
Gly Gly Glu Phe Phe Tyr Cys Asn Thr Thr Gl - #n Leu Phe Asn Ser Thr       
#       365                                                               
- tgg aat aat act gaa ggg aca aat agc act ga - #a gga aat agc aca atc     
1152                                                                      
Trp Asn Asn Thr Glu Gly Thr Asn Ser Thr Gl - #u Gly Asn Ser Thr Ile       
#   380                                                                   
- aca ctc cca tgc aga ata aaa caa att ata aa - #t atg tgg cag gaa gta     
1200                                                                      
Thr Leu Pro Cys Arg Ile Lys Gln Ile Ile As - #n Met Trp Gln Glu Val       
385                 3 - #90                 3 - #95                 4 -   
#00                                                                       
- gga aaa gca acg tat gcc cct ccc atc aga gg - #a cga att aga tgc ata     
1248                                                                      
Gly Lys Ala Thr Tyr Ala Pro Pro Ile Arg Gl - #y Arg Ile Arg Cys Ile       
#               415                                                       
- tca aat att aca gga ctg cta tta aca aga ga - #t ggt ggt agg aat gtc     
1296                                                                      
Ser Asn Ile Thr Gly Leu Leu Leu Thr Arg As - #p Gly Gly Arg Asn Val       
#           430                                                           
- aca aac aat acc gaa acc ttc aga cct gga gg - #a gga gac atg agg gac     
1344                                                                      
Thr Asn Asn Thr Glu Thr Phe Arg Pro Gly Gl - #y Gly Asp Met Arg Asp       
#       445                                                               
- aat tgg aga agt gaa tta tat aaa tat aaa gt - #a gta aaa gtt gaa cca     
1392                                                                      
Asn Trp Arg Ser Glu Leu Tyr Lys Tyr Lys Va - #l Val Lys Val Glu Pro       
#   460                                                                   
- tta gga ata gca ccc acc aag gca aag aga ag - #a gtg gtg cac aga gac     
1440                                                                      
Leu Gly Ile Ala Pro Thr Lys Ala Lys Arg Ar - #g Val Val His Arg Asp       
465                 4 - #70                 4 - #75                 4 -   
#80                                                                       
- aaa aga gca gca cta gga gcc ttg ttc ctt gg - #g ttc tta gga gca taa     
1488                                                                      
Lys Arg Ala Ala Leu Gly Ala Leu Phe Leu Gl - #y Phe Leu Gly Ala           
#               495                                                       
#     1499                                                                
Lys Leu Leu                                                               
- <210> SEQ ID NO 25                                                      
<211> LENGTH: 495                                                         
<212> TYPE: PRT                                                           
<213> ORGANISM: HIV                                                       
- <400> SEQUENCE: 25                                                      
- Glu Val Pro Val Trp Lys Glu Ala Thr Thr Th - #r Leu Phe Cys Ala Ser     
#                15                                                       
- Asp Ala Lys Ala Tyr Asp Thr Gly Val His As - #n Val Trp Ala Thr His     
#            30                                                           
- Ala Cys Val Pro Thr Asp Pro Asn Pro Gln Gl - #u Ile Glu Leu Val Asn     
#        45                                                               
- Val Thr Glu Asp Phe Asn Met Trp Lys Asn Ly - #s Met Val Asp Gln Met     
#    60                                                                   
- His Glu Asp Ile Ile Ser Leu Trp Asp Glu Se - #r Leu Lys Pro Cys Val     
#80                                                                       
- Lys Leu Thr Pro Leu Cys Val Thr Leu Asn Cy - #s Ser Asp Val Asn Asn     
#                95                                                       
- Ser Thr Asn Pro Asn Asp Thr Asn Thr Asn Se - #r Thr Asn Thr Thr Ser     
#           110                                                           
- Ser Thr Pro Thr Ala Thr Thr Ser Ser Glu Gl - #u Lys Met Glu Lys Gly     
#       125                                                               
- Glu Ile Lys Asn Cys Ser Phe Asn Ile Thr Th - #r His Met Lys Asp Lys     
#   140                                                                   
- Ala Gln Lys Glu Tyr Ala Leu Phe Tyr Lys Le - #u Asp Ile Val Pro Ile     
145                 1 - #50                 1 - #55                 1 -   
#60                                                                       
- Asp Asp Asn Asn Ala Ser Tyr Arg Leu Ile Se - #r Cys Asn Thr Ser Asp     
#               175                                                       
- Ile Thr Gln Ala Cys Pro Lys Val Thr Phe Gl - #u Pro Ile Pro Ile His     
#           190                                                           
- Tyr Cys Ala Pro Ala Gly Phe Ala Ile Leu Ly - #s Cys Lys Asp Lys Lys     
#       205                                                               
- Phe Asn Gly Thr Gly Pro Cys Ser Lys Val Se - #r Thr Val Gln Cys Thr     
#   220                                                                   
- His Gly Ile Arg Pro Val Val Ser Thr Gln Le - #u Leu Leu Asn Gly Ser     
225                 2 - #30                 2 - #35                 2 -   
#40                                                                       
- Leu Ala Glu Glu Glu Val Val Ile Arg Ser Va - #l Asn Phe Thr Asp Asn     
#               255                                                       
- Ala Lys Ile Ile Ile Val Gln Leu Lys Glu Pr - #o Val Ala Ile Asn Cys     
#           270                                                           
- Thr Arg Pro Asn Asn Asn Thr Arg Lys Gly Il - #e His Leu Gly Pro Gly     
#       285                                                               
- Ser Thr Phe Tyr Thr Thr Gly Glu Ile Ile Gl - #y Asp Ile Arg Lys Ala     
#   300                                                                   
- Tyr Cys Lys Ile Ser Lys Glu Lys Trp Asn As - #n Thr Leu Arg Gln Val     
305                 3 - #10                 3 - #15                 3 -   
#20                                                                       
- Val Lys Lys Leu Arg Glu Gln Phe Gly Asn Ly - #s Thr Ile Ile Phe Asn     
#               335                                                       
- Arg Ser Ser Gly Gly Asp Pro Glu Ile Val Me - #t His Ser Phe Asn Cys     
#           350                                                           
- Gly Gly Glu Phe Phe Tyr Cys Asn Thr Thr Gl - #n Leu Phe Asn Ser Thr     
#       365                                                               
- Trp Asn Asn Thr Glu Gly Thr Asn Ser Thr Gl - #u Gly Asn Ser Thr Ile     
#   380                                                                   
- Thr Leu Pro Cys Arg Ile Lys Gln Ile Ile As - #n Met Trp Gln Glu Val     
385                 3 - #90                 3 - #95                 4 -   
#00                                                                       
- Gly Lys Ala Thr Tyr Ala Pro Pro Ile Arg Gl - #y Arg Ile Arg Cys Ile     
#               415                                                       
- Ser Asn Ile Thr Gly Leu Leu Leu Thr Arg As - #p Gly Gly Arg Asn Val     
#           430                                                           
- Thr Asn Asn Thr Glu Thr Phe Arg Pro Gly Gl - #y Gly Asp Met Arg Asp     
#       445                                                               
- Asn Trp Arg Ser Glu Leu Tyr Lys Tyr Lys Va - #l Val Lys Val Glu Pro     
#   460                                                                   
- Leu Gly Ile Ala Pro Thr Lys Ala Lys Arg Ar - #g Val Val His Arg Asp     
465                 4 - #70                 4 - #75                 4 -   
#80                                                                       
- Lys Arg Ala Ala Leu Gly Ala Leu Phe Leu Gl - #y Phe Leu Gly Ala         
#               495                                                       
- <210> SEQ ID NO 26                                                      
<211> LENGTH: 3                                                           
<212> TYPE: PRT                                                           
<213> ORGANISM: HIV                                                       
- <400> SEQUENCE: 26                                                      
- Lys Leu Leu                                                             
 1                                                                        
- <210> SEQ ID NO 27                                                      
<211> LENGTH: 1499                                                        
<212> TYPE: DNA                                                           
<213> ORGANISM: HIV                                                       
<220> FEATURE:                                                            
<221> NAME/KEY: CDS                                                       
<222> LOCATION: (1)...(1497)                                              
<221> NAME/KEY: misc.sub.-- feature                                       
<222> LOCATION: (1)...(1499)                                              
<223> OTHER INFORMATION: n = A,T,C or G                                   
- <400> SEQUENCE: 27                                                      
- gag gta cct gta tgg aaa gaa gca acc act ac - #t cta ttt tgt gca tca     
  48                                                                      
Glu Val Pro Val Trp Lys Glu Ala Thr Thr Th - #r Leu Phe Cys Ala Ser       
#                 15                                                      
- gat gct aaa gca tat gac aca gag gtg cat aa - #t gtt tgg gcc aca cat     
  96                                                                      
Asp Ala Lys Ala Tyr Asp Thr Glu Val His As - #n Val Trp Ala Thr His       
#             30                                                          
- gcc tgt gta ccc aca gac ccc aac cca caa ga - #a ata gaa ttg gta aat     
 144                                                                      
Ala Cys Val Pro Thr Asp Pro Asn Pro Gln Gl - #u Ile Glu Leu Val Asn       
#         45                                                              
- gtg aca gaa gat ttt aac atg tgg aaa aat aa - #a atg gta gac cag atg     
 192                                                                      
Val Thr Glu Asp Phe Asn Met Trp Lys Asn Ly - #s Met Val Asp Gln Met       
#     60                                                                  
- cat gag gat ata atc agt tta tgg gat gaa ag - #c cta aag cca tgt gta     
 240                                                                      
His Glu Asp Ile Ile Ser Leu Trp Asp Glu Se - #r Leu Lys Pro Cys Val       
# 80                                                                      
- aag tta acc cca ctt tgt gtt act cta aac tg - #c agt gat gtg aac aat     
 288                                                                      
Lys Leu Thr Pro Leu Cys Val Thr Leu Asn Cy - #s Ser Asp Val Asn Asn       
#                 95                                                      
- tcc aca aat cct aat gat act aat act aat tc - #c act aat act act tcc     
 336                                                                      
Ser Thr Asn Pro Asn Asp Thr Asn Thr Asn Se - #r Thr Asn Thr Thr Ser       
#           110                                                           
- tct act cct acg gcc act act agt agc gag ga - #a aag atg gag aag gga     
 384                                                                      
Ser Thr Pro Thr Ala Thr Thr Ser Ser Glu Gl - #u Lys Met Glu Lys Gly       
#       125                                                               
- gaa ata aaa aac tgc tct ttc aat atc acc ac - #a cac atg aaa gat aag     
 432                                                                      
Glu Ile Lys Asn Cys Ser Phe Asn Ile Thr Th - #r His Met Lys Asp Lys       
#   140                                                                   
- gta cag aaa gaa tat gca ctt ttt tat aaa ct - #t gat ata gta cca ata     
 480                                                                      
Val Gln Lys Glu Tyr Ala Leu Phe Tyr Lys Le - #u Asp Ile Val Pro Ile       
145                 1 - #50                 1 - #55                 1 -   
#60                                                                       
- gat gat aat aat acc agc tat agg ttg ata ag - #t tgt aat acc tca gtc     
 528                                                                      
Asp Asp Asn Asn Thr Ser Tyr Arg Leu Ile Se - #r Cys Asn Thr Ser Val       
#               175                                                       
- att aca cag gcc tgt cca atg gtg acc ttt ga - #g cca att ccc ata cat     
 576                                                                      
Ile Thr Gln Ala Cys Pro Met Val Thr Phe Gl - #u Pro Ile Pro Ile His       
#           190                                                           
- tat tgt gcc ccg gct ggt ttt gcg att cta aa - #g tgt aaa gat aag aag     
 624                                                                      
Tyr Cys Ala Pro Ala Gly Phe Ala Ile Leu Ly - #s Cys Lys Asp Lys Lys       
#       205                                                               
- ttc aat gga aca gga cca tgt tca aag gtc ag - #c aca gta caa tgt aca     
 672                                                                      
Phe Asn Gly Thr Gly Pro Cys Ser Lys Val Se - #r Thr Val Gln Cys Thr       
#   220                                                                   
- cat gga att agg cca gta gta tca act caa ct - #g ttg tta aat ggc agt     
 720                                                                      
His Gly Ile Arg Pro Val Val Ser Thr Gln Le - #u Leu Leu Asn Gly Ser       
225                 2 - #30                 2 - #35                 2 -   
#40                                                                       
- ctt gca gaa gaa gaa gta gta att aga tct gt - #c aat ttc aca gac aat     
 768                                                                      
Leu Ala Glu Glu Glu Val Val Ile Arg Ser Va - #l Asn Phe Thr Asp Asn       
#               255                                                       
- gct aaa atc ata ata gta cag ctg aaa gaa cc - #t gta gca att aat tgt     
 816                                                                      
Ala Lys Ile Ile Ile Val Gln Leu Lys Glu Pr - #o Val Ala Ile Asn Cys       
#           270                                                           
- aca aga ccc aac aac aat aca aga aaa ggt at - #a cat cta gga cca ggg     
 864                                                                      
Thr Arg Pro Asn Asn Asn Thr Arg Lys Gly Il - #e His Leu Gly Pro Gly       
#       285                                                               
- agc aca ttt tat aca aca gga gaa ata ata gg - #a gac ata aga aaa gca     
 912                                                                      
Ser Thr Phe Tyr Thr Thr Gly Glu Ile Ile Gl - #y Asp Ile Arg Lys Ala       
#   300                                                                   
- tat tgc aag att agt aaa gaa aaa tgg aat aa - #c act tta aga cag gta     
 960                                                                      
Tyr Cys Lys Ile Ser Lys Glu Lys Trp Asn As - #n Thr Leu Arg Gln Val       
305                 3 - #10                 3 - #15                 3 -   
#20                                                                       
- gtt aaa aaa tta aga gaa caa ttt ggg aat aa - #a aca ata att ttt aat     
1008                                                                      
Val Lys Lys Leu Arg Glu Gln Phe Gly Asn Ly - #s Thr Ile Ile Phe Asn       
#               335                                                       
- cga tcc tca gga ggg gac cca gaa att gta at - #g cac agt ttt aac tgt     
1056                                                                      
Arg Ser Ser Gly Gly Asp Pro Glu Ile Val Me - #t His Ser Phe Asn Cys       
#           350                                                           
- gga ggg gag ttt ttc tac tgt aat aca aca ca - #a ctg ttt aat agt act     
1104                                                                      
Gly Gly Glu Phe Phe Tyr Cys Asn Thr Thr Gl - #n Leu Phe Asn Ser Thr       
#       365                                                               
- tgg aat aat act gaa ggg aca aat agc act ga - #a gga aat agc aca atc     
1152                                                                      
Trp Asn Asn Thr Glu Gly Thr Asn Ser Thr Gl - #u Gly Asn Ser Thr Ile       
#   380                                                                   
- aca ctc cca tgc aga ata aaa caa att ata aa - #t atg tgg cag gaa gta     
1200                                                                      
Thr Leu Pro Cys Arg Ile Lys Gln Ile Ile As - #n Met Trp Gln Glu Val       
385                 3 - #90                 3 - #95                 4 -   
#00                                                                       
- gga aaa gca acg tat gcc cct ccc atc aga gg - #a cga att aga tgc ata     
1248                                                                      
Gly Lys Ala Thr Tyr Ala Pro Pro Ile Arg Gl - #y Arg Ile Arg Cys Ile       
#               415                                                       
- tca aat att aca gga ctg cta tta aca aga ga - #t ggt ggt agg aat gtc     
1296                                                                      
Ser Asn Ile Thr Gly Leu Leu Leu Thr Arg As - #p Gly Gly Arg Asn Val       
#           430                                                           
- aca aac aat acc gan ncc ttc aga cct gga gg - #a gga gac atg agg gac     
1344                                                                      
Thr Asn Asn Thr Xaa Xaa Phe Arg Pro Gly Gl - #y Gly Asp Met Arg Asp       
#       445                                                               
- aat tgg aga agt gaa tta tat aaa tat aaa gt - #a gta aaa gtt gaa cca     
1392                                                                      
Asn Trp Arg Ser Glu Leu Tyr Lys Tyr Lys Va - #l Val Lys Val Glu Pro       
#   460                                                                   
- tta gga ata gca ccc acc aag gca aag aga ag - #a gtg gtg cac aga gac     
1440                                                                      
Leu Gly Ile Ala Pro Thr Lys Ala Lys Arg Ar - #g Val Val His Arg Asp       
465                 4 - #70                 4 - #75                 4 -   
#80                                                                       
- aaa aga gca gca cta gga gct ttg ttc ctt gg - #g ttc tta gga gca taa     
1488                                                                      
Lys Arg Ala Ala Leu Gly Ala Leu Phe Leu Gl - #y Phe Leu Gly Ala           
#               495                                                       
#     1499                                                                
Lys Leu Leu                                                               
- <210> SEQ ID NO 28                                                      
<211> LENGTH: 495                                                         
<212> TYPE: PRT                                                           
<213> ORGANISM: HIV                                                       
<220> FEATURE:                                                            
<221> NAME/KEY: VARIANT                                                   
<222> LOCATION: (1)...(495)                                               
<223> OTHER INFORMATION: Xaa = Any Amino Aci - #d                         
- <400> SEQUENCE: 28                                                      
- Glu Val Pro Val Trp Lys Glu Ala Thr Thr Th - #r Leu Phe Cys Ala Ser     
#                15                                                       
- Asp Ala Lys Ala Tyr Asp Thr Glu Val His As - #n Val Trp Ala Thr His     
#            30                                                           
- Ala Cys Val Pro Thr Asp Pro Asn Pro Gln Gl - #u Ile Glu Leu Val Asn     
#        45                                                               
- Val Thr Glu Asp Phe Asn Met Trp Lys Asn Ly - #s Met Val Asp Gln Met     
#    60                                                                   
- His Glu Asp Ile Ile Ser Leu Trp Asp Glu Se - #r Leu Lys Pro Cys Val     
#80                                                                       
- Lys Leu Thr Pro Leu Cys Val Thr Leu Asn Cy - #s Ser Asp Val Asn Asn     
#                95                                                       
- Ser Thr Asn Pro Asn Asp Thr Asn Thr Asn Se - #r Thr Asn Thr Thr Ser     
#           110                                                           
- Ser Thr Pro Thr Ala Thr Thr Ser Ser Glu Gl - #u Lys Met Glu Lys Gly     
#       125                                                               
- Glu Ile Lys Asn Cys Ser Phe Asn Ile Thr Th - #r His Met Lys Asp Lys     
#   140                                                                   
- Val Gln Lys Glu Tyr Ala Leu Phe Tyr Lys Le - #u Asp Ile Val Pro Ile     
145                 1 - #50                 1 - #55                 1 -   
#60                                                                       
- Asp Asp Asn Asn Thr Ser Tyr Arg Leu Ile Se - #r Cys Asn Thr Ser Val     
#               175                                                       
- Ile Thr Gln Ala Cys Pro Met Val Thr Phe Gl - #u Pro Ile Pro Ile His     
#           190                                                           
- Tyr Cys Ala Pro Ala Gly Phe Ala Ile Leu Ly - #s Cys Lys Asp Lys Lys     
#       205                                                               
- Phe Asn Gly Thr Gly Pro Cys Ser Lys Val Se - #r Thr Val Gln Cys Thr     
#   220                                                                   
- His Gly Ile Arg Pro Val Val Ser Thr Gln Le - #u Leu Leu Asn Gly Ser     
225                 2 - #30                 2 - #35                 2 -   
#40                                                                       
- Leu Ala Glu Glu Glu Val Val Ile Arg Ser Va - #l Asn Phe Thr Asp Asn     
#               255                                                       
- Ala Lys Ile Ile Ile Val Gln Leu Lys Glu Pr - #o Val Ala Ile Asn Cys     
#           270                                                           
- Thr Arg Pro Asn Asn Asn Thr Arg Lys Gly Il - #e His Leu Gly Pro Gly     
#       285                                                               
- Ser Thr Phe Tyr Thr Thr Gly Glu Ile Ile Gl - #y Asp Ile Arg Lys Ala     
#   300                                                                   
- Tyr Cys Lys Ile Ser Lys Glu Lys Trp Asn As - #n Thr Leu Arg Gln Val     
305                 3 - #10                 3 - #15                 3 -   
#20                                                                       
- Val Lys Lys Leu Arg Glu Gln Phe Gly Asn Ly - #s Thr Ile Ile Phe Asn     
#               335                                                       
- Arg Ser Ser Gly Gly Asp Pro Glu Ile Val Me - #t His Ser Phe Asn Cys     
#           350                                                           
- Gly Gly Glu Phe Phe Tyr Cys Asn Thr Thr Gl - #n Leu Phe Asn Ser Thr     
#       365                                                               
- Trp Asn Asn Thr Glu Gly Thr Asn Ser Thr Gl - #u Gly Asn Ser Thr Ile     
#   380                                                                   
- Thr Leu Pro Cys Arg Ile Lys Gln Ile Ile As - #n Met Trp Gln Glu Val     
385                 3 - #90                 3 - #95                 4 -   
#00                                                                       
- Gly Lys Ala Thr Tyr Ala Pro Pro Ile Arg Gl - #y Arg Ile Arg Cys Ile     
#               415                                                       
- Ser Asn Ile Thr Gly Leu Leu Leu Thr Arg As - #p Gly Gly Arg Asn Val     
#           430                                                           
- Thr Asn Asn Thr Xaa Xaa Phe Arg Pro Gly Gl - #y Gly Asp Met Arg Asp     
#       445                                                               
- Asn Trp Arg Ser Glu Leu Tyr Lys Tyr Lys Va - #l Val Lys Val Glu Pro     
#   460                                                                   
- Leu Gly Ile Ala Pro Thr Lys Ala Lys Arg Ar - #g Val Val His Arg Asp     
465                 4 - #70                 4 - #75                 4 -   
#80                                                                       
- Lys Arg Ala Ala Leu Gly Ala Leu Phe Leu Gl - #y Phe Leu Gly Ala         
#               495                                                       
- <210> SEQ ID NO 29                                                      
<211> LENGTH: 3                                                           
<212> TYPE: PRT                                                           
<213> ORGANISM: HIV                                                       
- <400> SEQUENCE: 29                                                      
- Lys Leu Leu                                                             
 1                                                                        
- <210> SEQ ID NO 30                                                      
<211> LENGTH: 1475                                                        
<212> TYPE: DNA                                                           
<213> ORGANISM: HIV                                                       
<220> FEATURE:                                                            
<221> NAME/KEY: CDS                                                       
<222> LOCATION: (2)...(1450)                                              
- <400> SEQUENCE: 30                                                      
#cta ttt tgt gca tca gat     49 aac aca act                               
  Val Pro Val Trp Lys Glu Ala Asn Thr T - #hr Leu Phe Cys Ala Ser Asp     
# 15                                                                      
- gct aaa gca tat gat aga gaa gta cat aat gt - #t tgg gca aca cat gcc     
  97                                                                      
Ala Lys Ala Tyr Asp Arg Glu Val His Asn Va - #l Trp Ala Thr His Ala       
#             30                                                          
- tgt gta ccc aca gac ccc aac cca caa gaa at - #a gta ttg gga aat gtg     
 145                                                                      
Cys Val Pro Thr Asp Pro Asn Pro Gln Glu Il - #e Val Leu Gly Asn Val       
#         45                                                              
- aca gaa aat ttt aac atg tgg aaa aat aac at - #g gta gaa caa atg cat     
 193                                                                      
Thr Glu Asn Phe Asn Met Trp Lys Asn Asn Me - #t Val Glu Gln Met His       
#     60                                                                  
- gag gat ata atc aat tta tgg gat caa agc tt - #a aag cca tgt gta aag     
 241                                                                      
Glu Asp Ile Ile Asn Leu Trp Asp Gln Ser Le - #u Lys Pro Cys Val Lys       
# 80                                                                      
- tta act cca ctc tgt gtt act tta aag tgc aa - #g gat ctg gag agg aat     
 289                                                                      
Leu Thr Pro Leu Cys Val Thr Leu Lys Cys Ly - #s Asp Leu Glu Arg Asn       
#                 95                                                      
- act acc tat aat agc act att acc aat aat ag - #t agt ttg gag gga cta     
 337                                                                      
Thr Thr Tyr Asn Ser Thr Ile Thr Asn Asn Se - #r Ser Leu Glu Gly Leu       
#           110                                                           
- aga gaa caa atg aca aac tgc tct ttc aac at - #c acc aca agt ata aga     
 385                                                                      
Arg Glu Gln Met Thr Asn Cys Ser Phe Asn Il - #e Thr Thr Ser Ile Arg       
#       125                                                               
- gat aag gtg cag aaa gaa tat gca ctt ttg ta - #t aaa ctt gat gta gta     
 433                                                                      
Asp Lys Val Gln Lys Glu Tyr Ala Leu Leu Ty - #r Lys Leu Asp Val Val       
#   140                                                                   
- cca ata gaa gaa gat gac aat act agc tat ag - #a ttg ata agt tgt aac     
 481                                                                      
Pro Ile Glu Glu Asp Asp Asn Thr Ser Tyr Ar - #g Leu Ile Ser Cys Asn       
145                 1 - #50                 1 - #55                 1 -   
#60                                                                       
- acc tca gtc att aca cag gct tgt cca aag ac - #a tcc ttt gag cca att     
 529                                                                      
Thr Ser Val Ile Thr Gln Ala Cys Pro Lys Th - #r Ser Phe Glu Pro Ile       
#               175                                                       
- ccc ata cat tat tgt gcc ccg gct ggt ttt gc - #g att cta aag tgt aat     
 577                                                                      
Pro Ile His Tyr Cys Ala Pro Ala Gly Phe Al - #a Ile Leu Lys Cys Asn       
#           190                                                           
- gat aag aag ttc aat gga aca gga cca tgt aa - #a aat gtc agc aca gta     
 625                                                                      
Asp Lys Lys Phe Asn Gly Thr Gly Pro Cys Ly - #s Asn Val Ser Thr Val       
#       205                                                               
- caa tgt aca cat gga att agg cca gta gta tc - #a act caa ctg ttg tta     
 673                                                                      
Gln Cys Thr His Gly Ile Arg Pro Val Val Se - #r Thr Gln Leu Leu Leu       
#   220                                                                   
- aat ggc agt cta gca gaa gaa gag gta gta at - #c aga tct gcc aat ttc     
 721                                                                      
Asn Gly Ser Leu Ala Glu Glu Glu Val Val Il - #e Arg Ser Ala Asn Phe       
225                 2 - #30                 2 - #35                 2 -   
#40                                                                       
- aca gac aat gct aaa acc ata ata gta cat ct - #a aat gaa act gta aaa     
 769                                                                      
Thr Asp Asn Ala Lys Thr Ile Ile Val His Le - #u Asn Glu Thr Val Lys       
#               255                                                       
- att aat tgt aca aga ctt ggc aac aat aca ag - #a aaa agt ata aat ata     
 817                                                                      
Ile Asn Cys Thr Arg Leu Gly Asn Asn Thr Ar - #g Lys Ser Ile Asn Ile       
#           270                                                           
- gga cca ggg aga gta ctc tat gca aca gga ga - #a ata ata gga gac ata     
 865                                                                      
Gly Pro Gly Arg Val Leu Tyr Ala Thr Gly Gl - #u Ile Ile Gly Asp Ile       
#       285                                                               
- aga caa gca cat tgt aac att agt aga gca ca - #a tgg aat aag act tta     
 913                                                                      
Arg Gln Ala His Cys Asn Ile Ser Arg Ala Gl - #n Trp Asn Lys Thr Leu       
#   300                                                                   
- gaa aag gta gtt gac aaa tta aga aaa caa tt - #t ggg gat aat aca aca     
 961                                                                      
Glu Lys Val Val Asp Lys Leu Arg Lys Gln Ph - #e Gly Asp Asn Thr Thr       
305                 3 - #10                 3 - #15                 3 -   
#20                                                                       
- ata gct ttt aat cga tcc tca gga ggg gac cc - #a gaa att gta atg cac     
1009                                                                      
Ile Ala Phe Asn Arg Ser Ser Gly Gly Asp Pr - #o Glu Ile Val Met His       
#               335                                                       
- act ttt aat tgt gga ggg gaa ttt ttc tac tg - #t aat aca aca caa ctg     
1057                                                                      
Thr Phe Asn Cys Gly Gly Glu Phe Phe Tyr Cy - #s Asn Thr Thr Gln Leu       
#           350                                                           
- ttt aat agt act tgg aat aat act tgg aag ga - #t cct aac agg agt gac     
1105                                                                      
Phe Asn Ser Thr Trp Asn Asn Thr Trp Lys As - #p Pro Asn Arg Ser Asp       
#       365                                                               
- aat atc aca ctc cca tgc aga ata aaa caa at - #t ata aac atg tgg cag     
1153                                                                      
Asn Ile Thr Leu Pro Cys Arg Ile Lys Gln Il - #e Ile Asn Met Trp Gln       
#   380                                                                   
- gaa gta gga aaa gca atg tac gcc cct ccc at - #c aga ggg gaa att aga     
1201                                                                      
Glu Val Gly Lys Ala Met Tyr Ala Pro Pro Il - #e Arg Gly Glu Ile Arg       
385                 3 - #90                 3 - #95                 4 -   
#00                                                                       
- tgt tca tca aat atc aca ggg ctg cta cta ac - #a aga gat ggt ggt aat     
1249                                                                      
Cys Ser Ser Asn Ile Thr Gly Leu Leu Leu Th - #r Arg Asp Gly Gly Asn       
#               415                                                       
- gac gat ggt aat gac acg acc aca aac agg ac - #c gag atc ttc aga cct     
1297                                                                      
Asp Asp Gly Asn Asp Thr Thr Thr Asn Arg Th - #r Glu Ile Phe Arg Pro       
#           430                                                           
- gga gga gga gat atg agg gac aat tgg aga ag - #t gaa tta tat aga tat     
1345                                                                      
Gly Gly Gly Asp Met Arg Asp Asn Trp Arg Se - #r Glu Leu Tyr Arg Tyr       
#       445                                                               
- aaa gta gta aaa att gaa cca tta gga ata gc - #a ccc acc agg gca aag     
1393                                                                      
Lys Val Val Lys Ile Glu Pro Leu Gly Ile Al - #a Pro Thr Arg Ala Lys       
#   460                                                                   
- aga aga gtg gtg cag aga gaa aaa aga gca gt - #a gga cta gga gct ttg     
1441                                                                      
Arg Arg Val Val Gln Arg Glu Lys Arg Ala Va - #l Gly Leu Gly Ala Leu       
465                 4 - #70                 4 - #75                 4 -   
#80                                                                       
#      1475        aggag cataaagctt ctaga                                 
Phe Leu Gly                                                               
- <210> SEQ ID NO 31                                                      
<211> LENGTH: 483                                                         
<212> TYPE: PRT                                                           
<213> ORGANISM: HIV                                                       
- <400> SEQUENCE: 31                                                      
- Val Pro Val Trp Lys Glu Ala Asn Thr Thr Le - #u Phe Cys Ala Ser Asp     
#                15                                                       
- Ala Lys Ala Tyr Asp Arg Glu Val His Asn Va - #l Trp Ala Thr His Ala     
#            30                                                           
- Cys Val Pro Thr Asp Pro Asn Pro Gln Glu Il - #e Val Leu Gly Asn Val     
#        45                                                               
- Thr Glu Asn Phe Asn Met Trp Lys Asn Asn Me - #t Val Glu Gln Met His     
#    60                                                                   
- Glu Asp Ile Ile Asn Leu Trp Asp Gln Ser Le - #u Lys Pro Cys Val Lys     
#80                                                                       
- Leu Thr Pro Leu Cys Val Thr Leu Lys Cys Ly - #s Asp Leu Glu Arg Asn     
#                95                                                       
- Thr Thr Tyr Asn Ser Thr Ile Thr Asn Asn Se - #r Ser Leu Glu Gly Leu     
#           110                                                           
- Arg Glu Gln Met Thr Asn Cys Ser Phe Asn Il - #e Thr Thr Ser Ile Arg     
#       125                                                               
- Asp Lys Val Gln Lys Glu Tyr Ala Leu Leu Ty - #r Lys Leu Asp Val Val     
#   140                                                                   
- Pro Ile Glu Glu Asp Asp Asn Thr Ser Tyr Ar - #g Leu Ile Ser Cys Asn     
145                 1 - #50                 1 - #55                 1 -   
#60                                                                       
- Thr Ser Val Ile Thr Gln Ala Cys Pro Lys Th - #r Ser Phe Glu Pro Ile     
#               175                                                       
- Pro Ile His Tyr Cys Ala Pro Ala Gly Phe Al - #a Ile Leu Lys Cys Asn     
#           190                                                           
- Asp Lys Lys Phe Asn Gly Thr Gly Pro Cys Ly - #s Asn Val Ser Thr Val     
#       205                                                               
- Gln Cys Thr His Gly Ile Arg Pro Val Val Se - #r Thr Gln Leu Leu Leu     
#   220                                                                   
- Asn Gly Ser Leu Ala Glu Glu Glu Val Val Il - #e Arg Ser Ala Asn Phe     
225                 2 - #30                 2 - #35                 2 -   
#40                                                                       
- Thr Asp Asn Ala Lys Thr Ile Ile Val His Le - #u Asn Glu Thr Val Lys     
#               255                                                       
- Ile Asn Cys Thr Arg Leu Gly Asn Asn Thr Ar - #g Lys Ser Ile Asn Ile     
#           270                                                           
- Gly Pro Gly Arg Val Leu Tyr Ala Thr Gly Gl - #u Ile Ile Gly Asp Ile     
#       285                                                               
- Arg Gln Ala His Cys Asn Ile Ser Arg Ala Gl - #n Trp Asn Lys Thr Leu     
#   300                                                                   
- Glu Lys Val Val Asp Lys Leu Arg Lys Gln Ph - #e Gly Asp Asn Thr Thr     
305                 3 - #10                 3 - #15                 3 -   
#20                                                                       
- Ile Ala Phe Asn Arg Ser Ser Gly Gly Asp Pr - #o Glu Ile Val Met His     
#               335                                                       
- Thr Phe Asn Cys Gly Gly Glu Phe Phe Tyr Cy - #s Asn Thr Thr Gln Leu     
#           350                                                           
- Phe Asn Ser Thr Trp Asn Asn Thr Trp Lys As - #p Pro Asn Arg Ser Asp     
#       365                                                               
- Asn Ile Thr Leu Pro Cys Arg Ile Lys Gln Il - #e Ile Asn Met Trp Gln     
#   380                                                                   
- Glu Val Gly Lys Ala Met Tyr Ala Pro Pro Il - #e Arg Gly Glu Ile Arg     
385                 3 - #90                 3 - #95                 4 -   
#00                                                                       
- Cys Ser Ser Asn Ile Thr Gly Leu Leu Leu Th - #r Arg Asp Gly Gly Asn     
#               415                                                       
- Asp Asp Gly Asn Asp Thr Thr Thr Asn Arg Th - #r Glu Ile Phe Arg Pro     
#           430                                                           
- Gly Gly Gly Asp Met Arg Asp Asn Trp Arg Se - #r Glu Leu Tyr Arg Tyr     
#       445                                                               
- Lys Val Val Lys Ile Glu Pro Leu Gly Ile Al - #a Pro Thr Arg Ala Lys     
#   460                                                                   
- Arg Arg Val Val Gln Arg Glu Lys Arg Ala Va - #l Gly Leu Gly Ala Leu     
465                 4 - #70                 4 - #75                 4 -   
#80                                                                       
- Phe Leu Gly                                                             
- <210> SEQ ID NO 32                                                      
<211> LENGTH: 1475                                                        
<212> TYPE: DNA                                                           
<213> ORGANISM: HIV                                                       
<220> FEATURE:                                                            
<221> NAME/KEY: CDS                                                       
<222> LOCATION: (2)...(1474)                                              
- <400> SEQUENCE: 32                                                      
#cta ttt tgt gca tca gat     49 aac aca act                               
  Val Pro Val Trp Lys Glu Ala Asn Thr T - #hr Leu Phe Cys Ala Ser Asp     
# 15                                                                      
- gct aaa gca tat gat aga gaa gta cat aat gt - #t tgg gca aca cat gcc     
  97                                                                      
Ala Lys Ala Tyr Asp Arg Glu Val His Asn Va - #l Trp Ala Thr His Ala       
#             30                                                          
- tgt gta ccc aca gac ccc aac cca caa gaa at - #a gta ttg gga aat gtg     
 145                                                                      
Cys Val Pro Thr Asp Pro Asn Pro Gln Glu Il - #e Val Leu Gly Asn Val       
#         45                                                              
- aca gaa aat ttt aac atg tgg aaa aat aac at - #g gta gaa caa atg cat     
 193                                                                      
Thr Glu Asn Phe Asn Met Trp Lys Asn Asn Me - #t Val Glu Gln Met His       
#     60                                                                  
- gag gat ata atc aat tta tgg gat caa agc tt - #a aag cca tgt gta aag     
 241                                                                      
Glu Asp Ile Ile Asn Leu Trp Asp Gln Ser Le - #u Lys Pro Cys Val Lys       
# 80                                                                      
- tta act cca ctc tgt gtt act tta aag tgc aa - #g gat ctg gag agg aat     
 289                                                                      
Leu Thr Pro Leu Cys Val Thr Leu Lys Cys Ly - #s Asp Leu Glu Arg Asn       
#                 95                                                      
- act acc tat aat agc act att acc aat aat ag - #t agt ttg gag gga cta     
 337                                                                      
Thr Thr Tyr Asn Ser Thr Ile Thr Asn Asn Se - #r Ser Leu Glu Gly Leu       
#           110                                                           
- aga gaa caa atg aca aac tgc tct ttc aac at - #c acc aca agt ata aga     
 385                                                                      
Arg Glu Gln Met Thr Asn Cys Ser Phe Asn Il - #e Thr Thr Ser Ile Arg       
#       125                                                               
- gat aag gtg cag aaa gaa tat gca ctt ttg ta - #t aaa ctt gat gta gta     
 433                                                                      
Asp Lys Val Gln Lys Glu Tyr Ala Leu Leu Ty - #r Lys Leu Asp Val Val       
#   140                                                                   
- cca ata gaa gaa gat gac aat act agc tat ag - #a ttg ata agt tgt aac     
 481                                                                      
Pro Ile Glu Glu Asp Asp Asn Thr Ser Tyr Ar - #g Leu Ile Ser Cys Asn       
145                 1 - #50                 1 - #55                 1 -   
#60                                                                       
- acc tca gtc att aca cag gct tgt cca aag ac - #a tcc ttt gag cca att     
 529                                                                      
Thr Ser Val Ile Thr Gln Ala Cys Pro Lys Th - #r Ser Phe Glu Pro Ile       
#               175                                                       
- ccc ata cat tat tgt gcc ccg gct ggt ttt gc - #g att cta aag tgt aat     
 577                                                                      
Pro Ile His Tyr Cys Ala Pro Ala Gly Phe Al - #a Ile Leu Lys Cys Asn       
#           190                                                           
- gat aag aag ttc aat gga aca gga cca tgt aa - #a aat gtc agc aca gta     
 625                                                                      
Asp Lys Lys Phe Asn Gly Thr Gly Pro Cys Ly - #s Asn Val Ser Thr Val       
#       205                                                               
- caa tgt aca cat gga att agg cca gta gta tc - #a act caa ctg ttg tta     
 673                                                                      
Gln Cys Thr His Gly Ile Arg Pro Val Val Se - #r Thr Gln Leu Leu Leu       
#   220                                                                   
- aat ggc agt cta gca gaa gaa gag gta gta at - #c aga tct gcc aat ttc     
 721                                                                      
Asn Gly Ser Leu Ala Glu Glu Glu Val Val Il - #e Arg Ser Ala Asn Phe       
225                 2 - #30                 2 - #35                 2 -   
#40                                                                       
- aca gac aat gct aaa acc ata ata gta cat ct - #a aat gaa act gta aaa     
 769                                                                      
Thr Asp Asn Ala Lys Thr Ile Ile Val His Le - #u Asn Glu Thr Val Lys       
#               255                                                       
- att aat tgt aca aga ctt ggc aac aat aca ag - #a aaa agt ata aat ata     
 817                                                                      
Ile Asn Cys Thr Arg Leu Gly Asn Asn Thr Ar - #g Lys Ser Ile Asn Ile       
#           270                                                           
- gga cca ggg aga gta ctc tat gca aca gga ga - #a ata ata gga gac ata     
 865                                                                      
Gly Pro Gly Arg Val Leu Tyr Ala Thr Gly Gl - #u Ile Ile Gly Asp Ile       
#       285                                                               
- aga caa gca cat tgt aac att agt aga gca ca - #a tgg aat aag act tta     
 913                                                                      
Arg Gln Ala His Cys Asn Ile Ser Arg Ala Gl - #n Trp Asn Lys Thr Leu       
#   300                                                                   
- gaa aag gta gtt gac aag tta aga aaa caa tt - #t ggg gat aat aca aca     
 961                                                                      
Glu Lys Val Val Asp Lys Leu Arg Lys Gln Ph - #e Gly Asp Asn Thr Thr       
305                 3 - #10                 3 - #15                 3 -   
#20                                                                       
- ata gct ttt aat cga tcc tca gga ggg gac cc - #a gaa att gta atg cac     
1009                                                                      
Ile Ala Phe Asn Arg Ser Ser Gly Gly Asp Pr - #o Glu Ile Val Met His       
#               335                                                       
- act ttt aat tgt gga ggg gaa ttt ttc tac tg - #t aat aca aca caa ctg     
1057                                                                      
Thr Phe Asn Cys Gly Gly Glu Phe Phe Tyr Cy - #s Asn Thr Thr Gln Leu       
#           350                                                           
- ttt aat agt act tgg aat aat act tgg aag ga - #t cct aac agg agt gac     
1105                                                                      
Phe Asn Ser Thr Trp Asn Asn Thr Trp Lys As - #p Pro Asn Arg Ser Asp       
#       365                                                               
- aat atc aca ctc cca tgc aga ata aaa caa at - #t ata aac atg tgg cag     
1153                                                                      
Asn Ile Thr Leu Pro Cys Arg Ile Lys Gln Il - #e Ile Asn Met Trp Gln       
#   380                                                                   
- gaa gta gga aaa gca atg tac gcc cct ccc at - #c aga ggg gaa att aga     
1201                                                                      
Glu Val Gly Lys Ala Met Tyr Ala Pro Pro Il - #e Arg Gly Glu Ile Arg       
385                 3 - #90                 3 - #95                 4 -   
#00                                                                       
- tgt tca tca aat atc aca ggg ctg cta cta ac - #a aga gat ggt ggt aat     
1249                                                                      
Cys Ser Ser Asn Ile Thr Gly Leu Leu Leu Th - #r Arg Asp Gly Gly Asn       
#               415                                                       
- gac gat ggt aat gac acg acc aca aac agg ac - #c gag atc ttc aga cct     
1297                                                                      
Asp Asp Gly Asn Asp Thr Thr Thr Asn Arg Th - #r Glu Ile Phe Arg Pro       
#           430                                                           
- gga gga gga gat atg agg gac aat tgg aga ag - #t gaa tta tat aga tat     
1345                                                                      
Gly Gly Gly Asp Met Arg Asp Asn Trp Arg Se - #r Glu Leu Tyr Arg Tyr       
#       445                                                               
- aaa gta gta aaa att gaa cca tta gga ata gc - #a ccc acc agg gca aag     
1393                                                                      
Lys Val Val Lys Ile Glu Pro Leu Gly Ile Al - #a Pro Thr Arg Ala Lys       
#   460                                                                   
- aga aga gtg gtg cag aga gaa aaa aga gca gt - #a gga cta gga gct ttg     
1441                                                                      
Arg Arg Val Val Gln Arg Glu Lys Arg Ala Va - #l Gly Leu Gly Ala Leu       
465                 4 - #70                 4 - #75                 4 -   
#80                                                                       
#      1475gg ttc ttg gga gca taa agc ttc ta - #g a                       
Phe Leu Gly Phe Leu Gly Ala  *  Ser Phe                                   
                485                                                       
- <210> SEQ ID NO 33                                                      
<211> LENGTH: 487                                                         
<212> TYPE: PRT                                                           
<213> ORGANISM: HIV                                                       
- <400> SEQUENCE: 33                                                      
- Val Pro Val Trp Lys Glu Ala Asn Thr Thr Le - #u Phe Cys Ala Ser Asp     
#                15                                                       
- Ala Lys Ala Tyr Asp Arg Glu Val His Asn Va - #l Trp Ala Thr His Ala     
#            30                                                           
- Cys Val Pro Thr Asp Pro Asn Pro Gln Glu Il - #e Val Leu Gly Asn Val     
#        45                                                               
- Thr Glu Asn Phe Asn Met Trp Lys Asn Asn Me - #t Val Glu Gln Met His     
#    60                                                                   
- Glu Asp Ile Ile Asn Leu Trp Asp Gln Ser Le - #u Lys Pro Cys Val Lys     
#80                                                                       
- Leu Thr Pro Leu Cys Val Thr Leu Lys Cys Ly - #s Asp Leu Glu Arg Asn     
#                95                                                       
- Thr Thr Tyr Asn Ser Thr Ile Thr Asn Asn Se - #r Ser Leu Glu Gly Leu     
#           110                                                           
- Arg Glu Gln Met Thr Asn Cys Ser Phe Asn Il - #e Thr Thr Ser Ile Arg     
#       125                                                               
- Asp Lys Val Gln Lys Glu Tyr Ala Leu Leu Ty - #r Lys Leu Asp Val Val     
#   140                                                                   
- Pro Ile Glu Glu Asp Asp Asn Thr Ser Tyr Ar - #g Leu Ile Ser Cys Asn     
145                 1 - #50                 1 - #55                 1 -   
#60                                                                       
- Thr Ser Val Ile Thr Gln Ala Cys Pro Lys Th - #r Ser Phe Glu Pro Ile     
#               175                                                       
- Pro Ile His Tyr Cys Ala Pro Ala Gly Phe Al - #a Ile Leu Lys Cys Asn     
#           190                                                           
- Asp Lys Lys Phe Asn Gly Thr Gly Pro Cys Ly - #s Asn Val Ser Thr Val     
#       205                                                               
- Gln Cys Thr His Gly Ile Arg Pro Val Val Se - #r Thr Gln Leu Leu Leu     
#   220                                                                   
- Asn Gly Ser Leu Ala Glu Glu Glu Val Val Il - #e Arg Ser Ala Asn Phe     
225                 2 - #30                 2 - #35                 2 -   
#40                                                                       
- Thr Asp Asn Ala Lys Thr Ile Ile Val His Le - #u Asn Glu Thr Val Lys     
#               255                                                       
- Ile Asn Cys Thr Arg Leu Gly Asn Asn Thr Ar - #g Lys Ser Ile Asn Ile     
#           270                                                           
- Gly Pro Gly Arg Val Leu Tyr Ala Thr Gly Gl - #u Ile Ile Gly Asp Ile     
#       285                                                               
- Arg Gln Ala His Cys Asn Ile Ser Arg Ala Gl - #n Trp Asn Lys Thr Leu     
#   300                                                                   
- Glu Lys Val Val Asp Lys Leu Arg Lys Gln Ph - #e Gly Asp Asn Thr Thr     
305                 3 - #10                 3 - #15                 3 -   
#20                                                                       
- Ile Ala Phe Asn Arg Ser Ser Gly Gly Asp Pr - #o Glu Ile Val Met His     
#               335                                                       
- Thr Phe Asn Cys Gly Gly Glu Phe Phe Tyr Cy - #s Asn Thr Thr Gln Leu     
#           350                                                           
- Phe Asn Ser Thr Trp Asn Asn Thr Trp Lys As - #p Pro Asn Arg Ser Asp     
#       365                                                               
- Asn Ile Thr Leu Pro Cys Arg Ile Lys Gln Il - #e Ile Asn Met Trp Gln     
#   380                                                                   
- Glu Val Gly Lys Ala Met Tyr Ala Pro Pro Il - #e Arg Gly Glu Ile Arg     
385                 3 - #90                 3 - #95                 4 -   
#00                                                                       
- Cys Ser Ser Asn Ile Thr Gly Leu Leu Leu Th - #r Arg Asp Gly Gly Asn     
#               415                                                       
- Asp Asp Gly Asn Asp Thr Thr Thr Asn Arg Th - #r Glu Ile Phe Arg Pro     
#           430                                                           
- Gly Gly Gly Asp Met Arg Asp Asn Trp Arg Se - #r Glu Leu Tyr Arg Tyr     
#       445                                                               
- Lys Val Val Lys Ile Glu Pro Leu Gly Ile Al - #a Pro Thr Arg Ala Lys     
#   460                                                                   
- Arg Arg Val Val Gln Arg Glu Lys Arg Ala Va - #l Gly Leu Gly Ala Leu     
465                 4 - #70                 4 - #75                 4 -   
#80                                                                       
- Phe Leu Gly Phe Leu Gly Ala                                             
                485                                                       
- <210> SEQ ID NO 34                                                      
<211> LENGTH: 2                                                           
<212> TYPE: PRT                                                           
<213> ORGANISM: HIV                                                       
- <400> SEQUENCE: 34                                                      
- Ser Phe                                                                 
 1                                                                        
- <210> SEQ ID NO 35                                                      
<211> LENGTH: 1435                                                        
<212> TYPE: DNA                                                           
<213> ORGANISM: HIV                                                       
<220> FEATURE:                                                            
<221> NAME/KEY: CDS                                                       
<222> LOCATION: (1)...(1434)                                              
- <400> SEQUENCE: 35                                                      
- ctc gag gta cct gtg tgg aaa gaa gca acc ac - #c act cta ttt tgt gca     
  48                                                                      
Leu Glu Val Pro Val Trp Lys Glu Ala Thr Th - #r Thr Leu Phe Cys Ala       
#                 15                                                      
- tca gat gct aaa gca tat gat tca gag gca ca - #t aat gtt tgg gcc aca     
  96                                                                      
Ser Asp Ala Lys Ala Tyr Asp Ser Glu Ala Hi - #s Asn Val Trp Ala Thr       
#             30                                                          
- cat gcc tgt gta ccc aca gac ccc aac cca ca - #a gaa gta gaa ttg gaa     
 144                                                                      
His Ala Cys Val Pro Thr Asp Pro Asn Pro Gl - #n Glu Val Glu Leu Glu       
#         45                                                              
- aat gtg aca gaa aat ttt aac atg tgg aaa aa - #t aac atg gta gaa cag     
 192                                                                      
Asn Val Thr Glu Asn Phe Asn Met Trp Lys As - #n Asn Met Val Glu Gln       
#     60                                                                  
- atg cat ggg gat ata att agt tta tgg gat ca - #a agc cta aag cca tgt     
 240                                                                      
Met His Gly Asp Ile Ile Ser Leu Trp Asp Gl - #n Ser Leu Lys Pro Cys       
# 80                                                                      
- gta aaa tta acc cca ctc tgt gtt acg tta aa - #t tgc act gac cca aat     
 288                                                                      
Val Lys Leu Thr Pro Leu Cys Val Thr Leu As - #n Cys Thr Asp Pro Asn       
#                 95                                                      
- gtt act aat agc gag aga acg ata gag ggg gg - #a gaa ata aaa aat tgc     
 336                                                                      
Val Thr Asn Ser Glu Arg Thr Ile Glu Gly Gl - #y Glu Ile Lys Asn Cys       
#           110                                                           
- tct ttc aat atc acc aca aac ata aga gat ag - #g ttt cag aaa gaa tat     
 384                                                                      
Ser Phe Asn Ile Thr Thr Asn Ile Arg Asp Ar - #g Phe Gln Lys Glu Tyr       
#       125                                                               
- gca ctt ttt tat aaa ctt gat gta ata cca tt - #a ggt aat gat aat act     
 432                                                                      
Ala Leu Phe Tyr Lys Leu Asp Val Ile Pro Le - #u Gly Asn Asp Asn Thr       
#   140                                                                   
- agc tat agg ttg ata agt tgt aac acc tca gt - #c att aca cag gcc tgt     
 480                                                                      
Ser Tyr Arg Leu Ile Ser Cys Asn Thr Ser Va - #l Ile Thr Gln Ala Cys       
145                 1 - #50                 1 - #55                 1 -   
#60                                                                       
- cca aag gta tcc ttt gag cca att ccc ata ca - #t tat tgt gcc ccg gct     
 528                                                                      
Pro Lys Val Ser Phe Glu Pro Ile Pro Ile Hi - #s Tyr Cys Ala Pro Ala       
#               175                                                       
- ggt ttt gcg att cta aag tgt aaa gat aag aa - #g ttc aat gga aca gga     
 576                                                                      
Gly Phe Ala Ile Leu Lys Cys Lys Asp Lys Ly - #s Phe Asn Gly Thr Gly       
#           190                                                           
- cca tgt aca aat gtc agc aca gta caa tgt ac - #a cat gga att aag cca     
 624                                                                      
Pro Cys Thr Asn Val Ser Thr Val Gln Cys Th - #r His Gly Ile Lys Pro       
#       205                                                               
- gta gta tca act caa ctg ttg tta aat ggc ag - #t cta gca gaa gaa gac     
 672                                                                      
Val Val Ser Thr Gln Leu Leu Leu Asn Gly Se - #r Leu Ala Glu Glu Asp       
#   220                                                                   
- ata gta att aga tcc gcc aat ctc aca gac aa - #t gct aaa aac ata ata     
 720                                                                      
Ile Val Ile Arg Ser Ala Asn Leu Thr Asp As - #n Ala Lys Asn Ile Ile       
225                 2 - #30                 2 - #35                 2 -   
#40                                                                       
- gta cag ctg aat gaa tct gta aca atg aat tg - #t aca aga ccc aac aac     
 768                                                                      
Val Gln Leu Asn Glu Ser Val Thr Met Asn Cy - #s Thr Arg Pro Asn Asn       
#               255                                                       
- aat aca atg aaa agt ata cat ata gga cca gg - #c aga gca ttt tat gca     
 816                                                                      
Asn Thr Met Lys Ser Ile His Ile Gly Pro Gl - #y Arg Ala Phe Tyr Ala       
#           270                                                           
- aca gga aac ata ata gga gat ata aga caa gc - #a cat tgt aac att agt     
 864                                                                      
Thr Gly Asn Ile Ile Gly Asp Ile Arg Gln Al - #a His Cys Asn Ile Ser       
#       285                                                               
- gga aca aaa tgg aat gac act ttg aaa aag at - #a gct ata aaa tta aga     
 912                                                                      
Gly Thr Lys Trp Asn Asp Thr Leu Lys Lys Il - #e Ala Ile Lys Leu Arg       
#   300                                                                   
- gaa caa ttt aat aag aca ata gtc ttt aat ca - #a tcc tca gga ggg gac     
 960                                                                      
Glu Gln Phe Asn Lys Thr Ile Val Phe Asn Gl - #n Ser Ser Gly Gly Asp       
305                 3 - #10                 3 - #15                 3 -   
#20                                                                       
- cca gaa att gca acg ctc agt ttt aat tgt gg - #a ggg gaa ttt ttc tac     
1008                                                                      
Pro Glu Ile Ala Thr Leu Ser Phe Asn Cys Gl - #y Gly Glu Phe Phe Tyr       
#               335                                                       
- tgt aat tca aca caa ctg ttt aat agt act tg - #g aat agt act ggg tca     
1056                                                                      
Cys Asn Ser Thr Gln Leu Phe Asn Ser Thr Tr - #p Asn Ser Thr Gly Ser       
#           350                                                           
- aat aac act aaa gga aat gac aca atc aca ct - #c cca tgc aga ata aga     
1104                                                                      
Asn Asn Thr Lys Gly Asn Asp Thr Ile Thr Le - #u Pro Cys Arg Ile Arg       
#       365                                                               
- caa att ata aac atg tgg cag aaa ata gga aa - #a gca atg tat gcc cct     
1152                                                                      
Gln Ile Ile Asn Met Trp Gln Lys Ile Gly Ly - #s Ala Met Tyr Ala Pro       
#   380                                                                   
- ccc atc aaa ggg caa att aga tgt tca tca aa - #t att aca ggg cta ata     
1200                                                                      
Pro Ile Lys Gly Gln Ile Arg Cys Ser Ser As - #n Ile Thr Gly Leu Ile       
385                 3 - #90                 3 - #95                 4 -   
#00                                                                       
- tta aca aga gat ggt ggt aac aac aac atg ag - #c aag acc acc gag acc     
1248                                                                      
Leu Thr Arg Asp Gly Gly Asn Asn Asn Met Se - #r Lys Thr Thr Glu Thr       
#               415                                                       
- ttc aga cct gga gga gga gat atg agg gac aa - #t tgg aga agt gaa tta     
1296                                                                      
Phe Arg Pro Gly Gly Gly Asp Met Arg Asp As - #n Trp Arg Ser Glu Leu       
#           430                                                           
- tat aaa tat aaa gta gta aaa att gaa cca tt - #a gga gta gca ccc acc     
1344                                                                      
Tyr Lys Tyr Lys Val Val Lys Ile Glu Pro Le - #u Gly Val Ala Pro Thr       
#       445                                                               
- agg gca aag aga aga gtg gtg cag aga gaa aa - #a aga gca gtg gga ata     
1392                                                                      
Arg Ala Lys Arg Arg Val Val Gln Arg Glu Ly - #s Arg Ala Val Gly Ile       
#   460                                                                   
- gga gct gtg ttc ctt ggg ttc ttg gga gca ta - #a agc ttc tag             
#1434                                                                     
#*  Ser Phe Phe Leu Gly Phe Leu Gly Ala                                   
#   475             4 - #70                                               
#             1435                                                        
- <210> SEQ ID NO 36                                                      
<211> LENGTH: 474                                                         
<212> TYPE: PRT                                                           
<213> ORGANISM: HIV                                                       
- <400> SEQUENCE: 36                                                      
- Leu Glu Val Pro Val Trp Lys Glu Ala Thr Th - #r Thr Leu Phe Cys Ala     
#                15                                                       
- Ser Asp Ala Lys Ala Tyr Asp Ser Glu Ala Hi - #s Asn Val Trp Ala Thr     
#            30                                                           
- His Ala Cys Val Pro Thr Asp Pro Asn Pro Gl - #n Glu Val Glu Leu Glu     
#        45                                                               
- Asn Val Thr Glu Asn Phe Asn Met Trp Lys As - #n Asn Met Val Glu Gln     
#    60                                                                   
- Met His Gly Asp Ile Ile Ser Leu Trp Asp Gl - #n Ser Leu Lys Pro Cys     
#80                                                                       
- Val Lys Leu Thr Pro Leu Cys Val Thr Leu As - #n Cys Thr Asp Pro Asn     
#                95                                                       
- Val Thr Asn Ser Glu Arg Thr Ile Glu Gly Gl - #y Glu Ile Lys Asn Cys     
#           110                                                           
- Ser Phe Asn Ile Thr Thr Asn Ile Arg Asp Ar - #g Phe Gln Lys Glu Tyr     
#       125                                                               
- Ala Leu Phe Tyr Lys Leu Asp Val Ile Pro Le - #u Gly Asn Asp Asn Thr     
#   140                                                                   
- Ser Tyr Arg Leu Ile Ser Cys Asn Thr Ser Va - #l Ile Thr Gln Ala Cys     
145                 1 - #50                 1 - #55                 1 -   
#60                                                                       
- Pro Lys Val Ser Phe Glu Pro Ile Pro Ile Hi - #s Tyr Cys Ala Pro Ala     
#               175                                                       
- Gly Phe Ala Ile Leu Lys Cys Lys Asp Lys Ly - #s Phe Asn Gly Thr Gly     
#           190                                                           
- Pro Cys Thr Asn Val Ser Thr Val Gln Cys Th - #r His Gly Ile Lys Pro     
#       205                                                               
- Val Val Ser Thr Gln Leu Leu Leu Asn Gly Se - #r Leu Ala Glu Glu Asp     
#   220                                                                   
- Ile Val Ile Arg Ser Ala Asn Leu Thr Asp As - #n Ala Lys Asn Ile Ile     
225                 2 - #30                 2 - #35                 2 -   
#40                                                                       
- Val Gln Leu Asn Glu Ser Val Thr Met Asn Cy - #s Thr Arg Pro Asn Asn     
#               255                                                       
- Asn Thr Met Lys Ser Ile His Ile Gly Pro Gl - #y Arg Ala Phe Tyr Ala     
#           270                                                           
- Thr Gly Asn Ile Ile Gly Asp Ile Arg Gln Al - #a His Cys Asn Ile Ser     
#       285                                                               
- Gly Thr Lys Trp Asn Asp Thr Leu Lys Lys Il - #e Ala Ile Lys Leu Arg     
#   300                                                                   
- Glu Gln Phe Asn Lys Thr Ile Val Phe Asn Gl - #n Ser Ser Gly Gly Asp     
305                 3 - #10                 3 - #15                 3 -   
#20                                                                       
- Pro Glu Ile Ala Thr Leu Ser Phe Asn Cys Gl - #y Gly Glu Phe Phe Tyr     
#               335                                                       
- Cys Asn Ser Thr Gln Leu Phe Asn Ser Thr Tr - #p Asn Ser Thr Gly Ser     
#           350                                                           
- Asn Asn Thr Lys Gly Asn Asp Thr Ile Thr Le - #u Pro Cys Arg Ile Arg     
#       365                                                               
- Gln Ile Ile Asn Met Trp Gln Lys Ile Gly Ly - #s Ala Met Tyr Ala Pro     
#   380                                                                   
- Pro Ile Lys Gly Gln Ile Arg Cys Ser Ser As - #n Ile Thr Gly Leu Ile     
385                 3 - #90                 3 - #95                 4 -   
#00                                                                       
- Leu Thr Arg Asp Gly Gly Asn Asn Asn Met Se - #r Lys Thr Thr Glu Thr     
#               415                                                       
- Phe Arg Pro Gly Gly Gly Asp Met Arg Asp As - #n Trp Arg Ser Glu Leu     
#           430                                                           
- Tyr Lys Tyr Lys Val Val Lys Ile Glu Pro Le - #u Gly Val Ala Pro Thr     
#       445                                                               
- Arg Ala Lys Arg Arg Val Val Gln Arg Glu Ly - #s Arg Ala Val Gly Ile     
#   460                                                                   
- Gly Ala Val Phe Leu Gly Phe Leu Gly Ala                                 
465                 4 - #70                                               
- <210> SEQ ID NO 37                                                      
<211> LENGTH: 2                                                           
<212> TYPE: PRT                                                           
<213> ORGANISM: HIV                                                       
- <400> SEQUENCE: 37                                                      
- Ser Phe                                                                 
 1                                                                        
- <210> SEQ ID NO 38                                                      
<211> LENGTH: 1435                                                        
<212> TYPE: DNA                                                           
<213> ORGANISM: HIV                                                       
<220> FEATURE:                                                            
<221> NAME/KEY: CDS                                                       
<222> LOCATION: (1)...(1434)                                              
- <400> SEQUENCE: 38                                                      
- ctc gag gta cct gtg tgg aaa gaa gca acc ac - #c act cta ttt tgt gca     
  48                                                                      
Leu Glu Val Pro Val Trp Lys Glu Ala Thr Th - #r Thr Leu Phe Cys Ala       
#                 15                                                      
- tca gat gct aaa gca tat gat tca gag gca ca - #t aat gtt tgg gcc aca     
  96                                                                      
Ser Asp Ala Lys Ala Tyr Asp Ser Glu Ala Hi - #s Asn Val Trp Ala Thr       
#             30                                                          
- cat gcc tgt gta ccc aca gac ccc aac cca ca - #a gaa gta gaa ttg gaa     
 144                                                                      
His Ala Cys Val Pro Thr Asp Pro Asn Pro Gl - #n Glu Val Glu Leu Glu       
#         45                                                              
- aat gtg aca gaa aat ttt aac atg tgg aaa aa - #t aac atg gta gaa cag     
 192                                                                      
Asn Val Thr Glu Asn Phe Asn Met Trp Lys As - #n Asn Met Val Glu Gln       
#     60                                                                  
- atg cat ggg gat ata att agt tta tgg gat ca - #a agc cta aag cca tgt     
 240                                                                      
Met His Gly Asp Ile Ile Ser Leu Trp Asp Gl - #n Ser Leu Lys Pro Cys       
# 80                                                                      
- gta aaa tta acc cca ctc tgt gtt acg tta aa - #t tgc act gac cca aat     
 288                                                                      
Val Lys Leu Thr Pro Leu Cys Val Thr Leu As - #n Cys Thr Asp Pro Asn       
#                 95                                                      
- gtt act aat agc gag aga acg ata gag ggg gg - #a gaa ata aaa aat tgc     
 336                                                                      
Val Thr Asn Ser Glu Arg Thr Ile Glu Gly Gl - #y Glu Ile Lys Asn Cys       
#           110                                                           
- tct ttc aat atc acc aca aac ata aga gat ag - #g ttt cag aaa gaa tat     
 384                                                                      
Ser Phe Asn Ile Thr Thr Asn Ile Arg Asp Ar - #g Phe Gln Lys Glu Tyr       
#       125                                                               
- gca ctt ttt tat aaa ctt gat gta ata cca tt - #a ggt aat gat aat act     
 432                                                                      
Ala Leu Phe Tyr Lys Leu Asp Val Ile Pro Le - #u Gly Asn Asp Asn Thr       
#   140                                                                   
- agc tat agg ttg ata agt tgt aac acc tca gt - #c att aca cag gcc tgt     
 480                                                                      
Ser Tyr Arg Leu Ile Ser Cys Asn Thr Ser Va - #l Ile Thr Gln Ala Cys       
145                 1 - #50                 1 - #55                 1 -   
#60                                                                       
- cca aag gta tcc ttt gag cca att ccc ata ca - #t tat tgt gcc ccg gct     
 528                                                                      
Pro Lys Val Ser Phe Glu Pro Ile Pro Ile Hi - #s Tyr Cys Ala Pro Ala       
#               175                                                       
- ggt ttt gcg att cta aag tgt aaa gat aag aa - #g ttc aat gga aca gga     
 576                                                                      
Gly Phe Ala Ile Leu Lys Cys Lys Asp Lys Ly - #s Phe Asn Gly Thr Gly       
#           190                                                           
- cca tgt aca aat gtc agc aca gta caa tgt ac - #a cat gga att aag cca     
 624                                                                      
Pro Cys Thr Asn Val Ser Thr Val Gln Cys Th - #r His Gly Ile Lys Pro       
#       205                                                               
- gta gta tca act caa ctg ttg tta aat ggc ag - #t cta gca gaa gaa gac     
 672                                                                      
Val Val Ser Thr Gln Leu Leu Leu Asn Gly Se - #r Leu Ala Glu Glu Asp       
#   220                                                                   
- ata gta att aga tcc gcc aat ctc aca gac aa - #t gct aaa aac ata ata     
 720                                                                      
Ile Val Ile Arg Ser Ala Asn Leu Thr Asp As - #n Ala Lys Asn Ile Ile       
225                 2 - #30                 2 - #35                 2 -   
#40                                                                       
- gta cag ctg aat gaa tct gta aca atg aat tg - #t aca aga ccc aac aac     
 768                                                                      
Val Gln Leu Asn Glu Ser Val Thr Met Asn Cy - #s Thr Arg Pro Asn Asn       
#               255                                                       
- aat aca atg aaa agt ata cat ata gga cca gg - #c aga gca ttt tat gca     
 816                                                                      
Asn Thr Met Lys Ser Ile His Ile Gly Pro Gl - #y Arg Ala Phe Tyr Ala       
#           270                                                           
- aca gga aac ata ata gga gat ata aga caa gc - #a cat tgt aac att agt     
 864                                                                      
Thr Gly Asn Ile Ile Gly Asp Ile Arg Gln Al - #a His Cys Asn Ile Ser       
#       285                                                               
- gga aca aaa tgg aat gac act ttg aaa aag at - #a gct ata aaa tta aga     
 912                                                                      
Gly Thr Lys Trp Asn Asp Thr Leu Lys Lys Il - #e Ala Ile Lys Leu Arg       
#   300                                                                   
- gaa caa ttt aat aag aca ata gtc ttt aat ca - #a tcc tca gga ggg gac     
 960                                                                      
Glu Gln Phe Asn Lys Thr Ile Val Phe Asn Gl - #n Ser Ser Gly Gly Asp       
305                 3 - #10                 3 - #15                 3 -   
#20                                                                       
- cca gaa att gca acg ctc agt ttt aat tgt gg - #a ggg gaa ttt ttc tac     
1008                                                                      
Pro Glu Ile Ala Thr Leu Ser Phe Asn Cys Gl - #y Gly Glu Phe Phe Tyr       
#               335                                                       
- tgt aat tca aca caa ctg ttt aat agt act tg - #g aat agt act ggg tca     
1056                                                                      
Cys Asn Ser Thr Gln Leu Phe Asn Ser Thr Tr - #p Asn Ser Thr Gly Ser       
#           350                                                           
- aat aac act aaa gga aat gac aca atc aca ct - #c cca tgc aga ata aga     
1104                                                                      
Asn Asn Thr Lys Gly Asn Asp Thr Ile Thr Le - #u Pro Cys Arg Ile Arg       
#       365                                                               
- caa att ata aac atg tgg cag aaa ata gga aa - #a gca atg tat gcc cct     
1152                                                                      
Gln Ile Ile Asn Met Trp Gln Lys Ile Gly Ly - #s Ala Met Tyr Ala Pro       
#   380                                                                   
- ccc atc aaa ggg caa att aga tgt tca tca aa - #t att aca ggg cta ata     
1200                                                                      
Pro Ile Lys Gly Gln Ile Arg Cys Ser Ser As - #n Ile Thr Gly Leu Ile       
385                 3 - #90                 3 - #95                 4 -   
#00                                                                       
- tta aca aga gat ggt ggt aac aac aac atg ag - #c aag acc acc gag acc     
1248                                                                      
Leu Thr Arg Asp Gly Gly Asn Asn Asn Met Se - #r Lys Thr Thr Glu Thr       
#               415                                                       
- ttc aga cct gga gga gga gat atg agg gac aa - #t tgg aga agt gaa tta     
1296                                                                      
Phe Arg Pro Gly Gly Gly Asp Met Arg Asp As - #n Trp Arg Ser Glu Leu       
#           430                                                           
- tat aaa tat aaa gta gta aaa att gaa cca tt - #a gga gta gca ccc acc     
1344                                                                      
Tyr Lys Tyr Lys Val Val Lys Ile Glu Pro Le - #u Gly Val Ala Pro Thr       
#       445                                                               
- agg gca aag aga aga gtg gtg cag aga gaa aa - #a aga gca gtg gga ata     
1392                                                                      
Arg Ala Lys Arg Arg Val Val Gln Arg Glu Ly - #s Arg Ala Val Gly Ile       
#   460                                                                   
- gga gct gtg ttc ctt ggg ttc ttg gga gca ta - #a agc ttc tag             
#1434                                                                     
#*  Ser Phe Phe Leu Gly Phe Leu Gly Ala                                   
#   475             4 - #70                                               
#             1435                                                        
- <210> SEQ ID NO 39                                                      
<211> LENGTH: 474                                                         
<212> TYPE: PRT                                                           
<213> ORGANISM: HIV                                                       
- <400> SEQUENCE: 39                                                      
- Leu Glu Val Pro Val Trp Lys Glu Ala Thr Th - #r Thr Leu Phe Cys Ala     
#                15                                                       
- Ser Asp Ala Lys Ala Tyr Asp Ser Glu Ala Hi - #s Asn Val Trp Ala Thr     
#            30                                                           
- His Ala Cys Val Pro Thr Asp Pro Asn Pro Gl - #n Glu Val Glu Leu Glu     
#        45                                                               
- Asn Val Thr Glu Asn Phe Asn Met Trp Lys As - #n Asn Met Val Glu Gln     
#    60                                                                   
- Met His Gly Asp Ile Ile Ser Leu Trp Asp Gl - #n Ser Leu Lys Pro Cys     
#80                                                                       
- Val Lys Leu Thr Pro Leu Cys Val Thr Leu As - #n Cys Thr Asp Pro Asn     
#                95                                                       
- Val Thr Asn Ser Glu Arg Thr Ile Glu Gly Gl - #y Glu Ile Lys Asn Cys     
#           110                                                           
- Ser Phe Asn Ile Thr Thr Asn Ile Arg Asp Ar - #g Phe Gln Lys Glu Tyr     
#       125                                                               
- Ala Leu Phe Tyr Lys Leu Asp Val Ile Pro Le - #u Gly Asn Asp Asn Thr     
#   140                                                                   
- Ser Tyr Arg Leu Ile Ser Cys Asn Thr Ser Va - #l Ile Thr Gln Ala Cys     
145                 1 - #50                 1 - #55                 1 -   
#60                                                                       
- Pro Lys Val Ser Phe Glu Pro Ile Pro Ile Hi - #s Tyr Cys Ala Pro Ala     
#               175                                                       
- Gly Phe Ala Ile Leu Lys Cys Lys Asp Lys Ly - #s Phe Asn Gly Thr Gly     
#           190                                                           
- Pro Cys Thr Asn Val Ser Thr Val Gln Cys Th - #r His Gly Ile Lys Pro     
#       205                                                               
- Val Val Ser Thr Gln Leu Leu Leu Asn Gly Se - #r Leu Ala Glu Glu Asp     
#   220                                                                   
- Ile Val Ile Arg Ser Ala Asn Leu Thr Asp As - #n Ala Lys Asn Ile Ile     
225                 2 - #30                 2 - #35                 2 -   
#40                                                                       
- Val Gln Leu Asn Glu Ser Val Thr Met Asn Cy - #s Thr Arg Pro Asn Asn     
#               255                                                       
- Asn Thr Met Lys Ser Ile His Ile Gly Pro Gl - #y Arg Ala Phe Tyr Ala     
#           270                                                           
- Thr Gly Asn Ile Ile Gly Asp Ile Arg Gln Al - #a His Cys Asn Ile Ser     
#       285                                                               
- Gly Thr Lys Trp Asn Asp Thr Leu Lys Lys Il - #e Ala Ile Lys Leu Arg     
#   300                                                                   
- Glu Gln Phe Asn Lys Thr Ile Val Phe Asn Gl - #n Ser Ser Gly Gly Asp     
305                 3 - #10                 3 - #15                 3 -   
#20                                                                       
- Pro Glu Ile Ala Thr Leu Ser Phe Asn Cys Gl - #y Gly Glu Phe Phe Tyr     
#               335                                                       
- Cys Asn Ser Thr Gln Leu Phe Asn Ser Thr Tr - #p Asn Ser Thr Gly Ser     
#           350                                                           
- Asn Asn Thr Lys Gly Asn Asp Thr Ile Thr Le - #u Pro Cys Arg Ile Arg     
#       365                                                               
- Gln Ile Ile Asn Met Trp Gln Lys Ile Gly Ly - #s Ala Met Tyr Ala Pro     
#   380                                                                   
- Pro Ile Lys Gly Gln Ile Arg Cys Ser Ser As - #n Ile Thr Gly Leu Ile     
385                 3 - #90                 3 - #95                 4 -   
#00                                                                       
- Leu Thr Arg Asp Gly Gly Asn Asn Asn Met Se - #r Lys Thr Thr Glu Thr     
#               415                                                       
- Phe Arg Pro Gly Gly Gly Asp Met Arg Asp As - #n Trp Arg Ser Glu Leu     
#           430                                                           
- Tyr Lys Tyr Lys Val Val Lys Ile Glu Pro Le - #u Gly Val Ala Pro Thr     
#       445                                                               
- Arg Ala Lys Arg Arg Val Val Gln Arg Glu Ly - #s Arg Ala Val Gly Ile     
#   460                                                                   
- Gly Ala Val Phe Leu Gly Phe Leu Gly Ala                                 
465                 4 - #70                                               
- <210> SEQ ID NO 40                                                      
<211> LENGTH: 2                                                           
<212> TYPE: PRT                                                           
<213> ORGANISM: HIV                                                       
- <400> SEQUENCE: 40                                                      
- Ser Phe                                                                 
 1                                                                        
- <210> SEQ ID NO 41                                                      
<211> LENGTH: 511                                                         
<212> TYPE: PRT                                                           
<213> ORGANISM: HIV                                                       
- <400> SEQUENCE: 41                                                      
- Met Arg Val Lys Gly Ile Arg Arg Asn Tyr Gl - #n His Trp Trp Gly Arg     
#                15                                                       
- Gly Thr Met Leu Leu Gly Leu Leu Met Ile Cy - #s Ser Ala Thr Glu Lys     
#            30                                                           
- Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Va - #l Trp Lys Glu Ala Thr     
#        45                                                               
- Thr Thr Leu Phe Cys Ala Ser Asp Ala Lys Al - #a Tyr Asp Thr Glu Ala     
#    60                                                                   
- His Asn Val Trp Ala Thr His Ala Cys Val Pr - #o Thr Asp Pro Asn Pro     
#80                                                                       
- Gln Glu Val Glu Leu Val Asn Val Thr Glu As - #n Phe Asn Met Trp Lys     
#                95                                                       
- Asn Asn Met Val Glu Gln Met His Glu Asp Il - #e Ile Ser Leu Trp Asn     
#           110                                                           
- Gln Ser Leu Lys Pro Cys Val Lys Leu Thr Pr - #o Leu Cys Val Thr Leu     
#       125                                                               
- Asn Cys Thr Asp Leu Arg Asn Thr Thr Asn Th - #r Asn Asn Ser Thr Asp     
#   140                                                                   
- Asn Asn Asn Ser Lys Ser Glu Gly Thr Ile Ly - #s Gly Gly Glu Met Lys     
145                 1 - #50                 1 - #55                 1 -   
#60                                                                       
- Asn Cys Ser Phe Asn Ile Thr Thr Ser Ile Gl - #y Asp Lys Met Gln Lys     
#               175                                                       
- Glu Tyr Ala Leu Leu Tyr Lys Leu Asp Ile Gl - #u Pro Ile Asp Asn Asp     
#           190                                                           
- Ser Thr Ser Tyr Arg Leu Ile Ser Cys Asn Th - #r Ser Val Ile Thr Gln     
#       205                                                               
- Ala Cys Pro Lys Ile Ser Phe Glu Pro Ile Pr - #o Ile His Tyr Cys Ala     
#   220                                                                   
- Pro Ala Gly Phe Ala Ile Leu Lys Cys Asn As - #p Lys Lys Phe Ser Gly     
225                 2 - #30                 2 - #35                 2 -   
#40                                                                       
- Lys Gly Ser Cys Lys Asn Val Ser Thr Val Gl - #n Cys Thr His Gly Ile     
#               255                                                       
- Arg Pro Val Val Ser Thr Gln Leu Leu Leu As - #n Gly Ser Leu Ala Glu     
#           270                                                           
- Glu Glu Val Val Ile Arg Ser Glu Asp Phe Th - #r Asp Asn Ala Lys Thr     
#       285                                                               
- Ile Ile Val His Leu Lys Glu Ser Val Gln Il - #e Asn Cys Thr Arg Pro     
#   300                                                                   
- Asn Tyr Asn Lys Arg Lys Arg Ile His Ile Gl - #y Pro Gly Arg Ala Phe     
305                 3 - #10                 3 - #15                 3 -   
#20                                                                       
- Tyr Thr Thr Lys Asn Ile Lys Gly Thr Ile Ar - #g Gln Ala His Cys Ile     
#               335                                                       
- Ile Ser Arg Ala Lys Trp Asn Asp Thr Leu Ar - #g Gln Ile Val Ser Lys     
#           350                                                           
- Leu Lys Glu Gln Phe Lys Asn Lys Thr Ile Va - #l Phe Asn Pro Ser Ser     
#       365                                                               
- Gly Gly Asp Pro Glu Ile Val Met His Ser Ph - #e Asn Cys Gly Gly Glu     
#   380                                                                   
- Phe Phe Tyr Cys Asn Thr Ser Pro Leu Phe As - #n Ser Ile Trp Asn Gly     
385                 3 - #90                 3 - #95                 4 -   
#00                                                                       
- Asn Asn Thr Trp Asn Asn Thr Thr Gly Ser As - #n Asn Asn Ile Thr Leu     
#               415                                                       
- Gln Cys Lys Ile Lys Gln Ile Ile Asn Met Tr - #p Gln Lys Val Gly Lys     
#           430                                                           
- Ala Met Tyr Ala Pro Pro Ile Glu Gly Gln Il - #e Arg Cys Ser Ser Asn     
#       445                                                               
- Ile Thr Gly Leu Leu Leu Thr Arg Asp Gly Gl - #y Glu Asp Thr Asp Thr     
#   460                                                                   
- Asn Asp Thr Glu Ile Phe Arg Pro Gly Gly Gl - #y Asp Met Arg Asp Asn     
465                 4 - #70                 4 - #75                 4 -   
#80                                                                       
- Trp Arg Ser Glu Leu Tyr Lys Tyr Lys Val Va - #l Thr Ile Glu Pro Leu     
#               495                                                       
- Gly Val Ala Pro Thr Lys Ala Lys Arg Arg Va - #l Val Gln Arg Glu         
#           510                                                           
- <210> SEQ ID NO 42                                                      
<211> LENGTH: 2800                                                        
<212> TYPE: DNA                                                           
<213> ORGANISM: HIV                                                       
<220> FEATURE:                                                            
<221> NAME/KEY: CDS                                                       
<222> LOCATION: (683)...(2419)                                            
- <400> SEQUENCE: 42                                                      
- ttcgagctcg cccgacattg attattgact agagtcgatc gacagctgtg ga - #atgtgtgt   
  60                                                                      
- cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aa - #gcatgcat   
 120                                                                      
- ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg ca - #gaagtatg   
 180                                                                      
- caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gc - #ccatcccg   
 240                                                                      
- cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tt - #tttttatt   
 300                                                                      
- tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg ag - #gaggcttt   
 360                                                                      
- tttggaggcc taggcttttg caaaaagcta gcttatccgg ccgggaacgg tg - #cattggaa   
 420                                                                      
- cgcggattcc ccgtgccaag agtcaggtaa gtaccgccta tagagtctat ag - #gcccaccc   
 480                                                                      
- ccttggcttc gttagaacgc ggctacaatt aatacataac cttttggatc ga - #tcctactg   
 540                                                                      
- acactgacat ccactttttc tttttctcca caggtgtcca ctcccaggtc ca - #actgcacc   
 600                                                                      
- tcggttcgcg aagctagctt gggctgcatc gattgaattc cactgccttc ca - #ccaagctc   
 660                                                                      
- tgcaggatcc cagagtcagg gg tct gta tct tcc tgc tgg - # tgg ctc cag ttc    
 712                                                                      
#       Ser Val Ser Ser Cys Trp T - #rp Leu Gln Phe                       
#      10                                                                 
- agg aac agt aaa ccc tgc tcc gaa tat tgc ct - #c tca cat ctc gtc aat     
 760                                                                      
Arg Asn Ser Lys Pro Cys Ser Glu Tyr Cys Le - #u Ser His Leu Val Asn       
#                 25                                                      
- ctc cgc gag gac tgg gga ccc tct gac aag ct - #t cag cgc gaa cga cca     
 808                                                                      
Leu Arg Glu Asp Trp Gly Pro Ser Asp Lys Le - #u Gln Arg Glu Arg Pro       
#             40                                                          
- act acc ccg atc atc agt tat cct taa ggt ct - #c ttt tgt gtg gtg cgt     
 856                                                                      
Thr Thr Pro Ile Ile Ser Tyr Pro  *  Gly - # Leu Phe Cys Val Val Arg       
#        55                                                               
- tcc ggt atg ggg ggg act gcc gcc agg ttg gg - #g gcc gtg att ttg ttt     
 904                                                                      
Ser Gly Met Gly Gly Thr Ala Ala Arg Leu Gl - #y Ala Val Ile Leu Phe       
#    70                                                                   
- gtc gtc ata gtg ggc ctc cat ggg gtc cgc gg - #c aaa tat gcc ttg gcg     
 952                                                                      
Val Val Ile Val Gly Leu His Gly Val Arg Gl - #y Lys Tyr Ala Leu Ala       
#90                                                                       
- gat gcc tct ctc aag atg gcc gac ccc aat cg - #a ttt cgc ggc aaa gac     
1000                                                                      
Asp Ala Ser Leu Lys Met Ala Asp Pro Asn Ar - #g Phe Arg Gly Lys Asp       
#                105                                                      
- ctt ccg gtc ctg gac cag ctg ctc gag gta cc - #t gtg tgg aaa gaa gca     
1048                                                                      
Leu Pro Val Leu Asp Gln Leu Leu Glu Val Pr - #o Val Trp Lys Glu Ala       
#           120                                                           
- aac acc act cta ttt tgt gca tca gat gct aa - #a gca tat aag aca gag     
1096                                                                      
Asn Thr Thr Leu Phe Cys Ala Ser Asp Ala Ly - #s Ala Tyr Lys Thr Glu       
#       135                                                               
- gca cat aat gtt tgg gcc aca cat gcc tgt gt - #a ccc aca gac ccc aaa     
1144                                                                      
Ala His Asn Val Trp Ala Thr His Ala Cys Va - #l Pro Thr Asp Pro Lys       
#   150                                                                   
- cca caa gaa ata aaa ttg gaa aat gtg aca ga - #a aat ttt aac atg tgg     
1192                                                                      
Pro Gln Glu Ile Lys Leu Glu Asn Val Thr Gl - #u Asn Phe Asn Met Trp       
155                 1 - #60                 1 - #65                 1 -   
#70                                                                       
- aaa aat aac atg gta gaa cag atg cat gag ga - #t ata atc agt tta tgg     
1240                                                                      
Lys Asn Asn Met Val Glu Gln Met His Glu As - #p Ile Ile Ser Leu Trp       
#               185                                                       
- gat caa agc cta aag cca tgt gta aaa tta ac - #c cca ctc tgt gtt act     
1288                                                                      
Asp Gln Ser Leu Lys Pro Cys Val Lys Leu Th - #r Pro Leu Cys Val Thr       
#           200                                                           
- tta aat tgc act gat ttg agg aat aat act aa - #t acc aat agt acc tac     
1336                                                                      
Leu Asn Cys Thr Asp Leu Arg Asn Asn Thr As - #n Thr Asn Ser Thr Tyr       
#       215                                                               
- gga aaa ata atg gag gga gga gag ata aaa aa - #c tgc tct ttc aat atc     
1384                                                                      
Gly Lys Ile Met Glu Gly Gly Glu Ile Lys As - #n Cys Ser Phe Asn Ile       
#   230                                                                   
- acc aca agc ata aaa gat aag ctg aaa gat at - #g tca ctt ttt tat aaa     
1432                                                                      
Thr Thr Ser Ile Lys Asp Lys Leu Lys Asp Me - #t Ser Leu Phe Tyr Lys       
235                 2 - #40                 2 - #45                 2 -   
#50                                                                       
- ctt gat gta gta cca ata ggt aat aat agt aa - #t act act agt tat agg     
1480                                                                      
Leu Asp Val Val Pro Ile Gly Asn Asn Ser As - #n Thr Thr Ser Tyr Arg       
#               265                                                       
- ttg ata agt tgt aac acc tca gtc att aca ca - #a gcc tgt cca aag aca     
1528                                                                      
Leu Ile Ser Cys Asn Thr Ser Val Ile Thr Gl - #n Ala Cys Pro Lys Thr       
#           280                                                           
- tcc ttt gag cca att ccc ata cat tat tgt gc - #c ccg gct ggt ttt gcg     
1576                                                                      
Ser Phe Glu Pro Ile Pro Ile His Tyr Cys Al - #a Pro Ala Gly Phe Ala       
#       295                                                               
- att ctc aag tgt aat gat aat aag ttc aat gg - #a aca gga cca tgt cca     
1624                                                                      
Ile Leu Lys Cys Asn Asp Asn Lys Phe Asn Gl - #y Thr Gly Pro Cys Pro       
#   310                                                                   
- aat gtc agc aca gta caa tgt aca cat gga at - #t agg cca gta gta tca     
1672                                                                      
Asn Val Ser Thr Val Gln Cys Thr His Gly Il - #e Arg Pro Val Val Ser       
315                 3 - #20                 3 - #25                 3 -   
#30                                                                       
- act caa ctg ctg tta aat ggc agt cta gca ga - #a aaa gag gta gtc ctt     
1720                                                                      
Thr Gln Leu Leu Leu Asn Gly Ser Leu Ala Gl - #u Lys Glu Val Val Leu       
#               345                                                       
- aga tct gaa aat ttc acg gac aat gct aaa ac - #c ata ata gta cag ctg     
1768                                                                      
Arg Ser Glu Asn Phe Thr Asp Asn Ala Lys Th - #r Ile Ile Val Gln Leu       
#           360                                                           
- aac gaa tct gta ata att gat tgt atg aga cc - #c aac aac aat aca aga     
1816                                                                      
Asn Glu Ser Val Ile Ile Asp Cys Met Arg Pr - #o Asn Asn Asn Thr Arg       
#       375                                                               
- aca agt ata cct atg gga cca ggg aaa gca tt - #t tat gca aca gga gat     
1864                                                                      
Thr Ser Ile Pro Met Gly Pro Gly Lys Ala Ph - #e Tyr Ala Thr Gly Asp       
#   390                                                                   
- gta ata gga gat ata aga cga gca cat tgt aa - #c att agt aga gca gga     
1912                                                                      
Val Ile Gly Asp Ile Arg Arg Ala His Cys As - #n Ile Ser Arg Ala Gly       
395                 4 - #00                 4 - #05                 4 -   
#10                                                                       
- tgg aat acc act tta caa cag ata gct aaa aa - #a tta aga gaa aaa ttt     
1960                                                                      
Trp Asn Thr Thr Leu Gln Gln Ile Ala Lys Ly - #s Leu Arg Glu Lys Phe       
#               425                                                       
- gag aac aaa aca ata gtt ttt aat cac tcc tc - #a gga ggg gac cca gaa     
2008                                                                      
Glu Asn Lys Thr Ile Val Phe Asn His Ser Se - #r Gly Gly Asp Pro Glu       
#           440                                                           
- att gta atg cac act ttt aat tgt gga ggg ga - #a ttt ttc tgc tgt aat     
2056                                                                      
Ile Val Met His Thr Phe Asn Cys Gly Gly Gl - #u Phe Phe Cys Cys Asn       
#       455                                                               
- tca aca cca ctg ttt aat agt act tgg aat ga - #t gca caa ctg ttt aat     
2104                                                                      
Ser Thr Pro Leu Phe Asn Ser Thr Trp Asn As - #p Ala Gln Leu Phe Asn       
#   470                                                                   
- agt act tgg gat gat act aaa tgg tca aaa gg - #c act aac gaa aat gac     
2152                                                                      
Ser Thr Trp Asp Asp Thr Lys Trp Ser Lys Gl - #y Thr Asn Glu Asn Asp       
475                 4 - #80                 4 - #85                 4 -   
#90                                                                       
- aca atc acc ctc cat tgc aga ata aaa caa at - #t ata aat atg tgg cag     
2200                                                                      
Thr Ile Thr Leu His Cys Arg Ile Lys Gln Il - #e Ile Asn Met Trp Gln       
#               505                                                       
- gaa gta gga aaa gca atg tat gcc cct ccc at - #c aaa gga caa att aga     
2248                                                                      
Glu Val Gly Lys Ala Met Tyr Ala Pro Pro Il - #e Lys Gly Gln Ile Arg       
#           520                                                           
- tgt gaa tca aat att aca ggg ctg cta tta ac - #a aga gat ggt ggt aac     
2296                                                                      
Cys Glu Ser Asn Ile Thr Gly Leu Leu Leu Th - #r Arg Asp Gly Gly Asn       
#       535                                                               
- gac acg agc aag aat aac act gag att ttc ag - #a cct gga gga gga aat     
2344                                                                      
Asp Thr Ser Lys Asn Asn Thr Glu Ile Phe Ar - #g Pro Gly Gly Gly Asn       
#   550                                                                   
- atg aag gac aat tgg aga agt gaa tta tat aa - #a tat aaa gta ata aaa     
2392                                                                      
Met Lys Asp Asn Trp Arg Ser Glu Leu Tyr Ly - #s Tyr Lys Val Ile Lys       
555                 5 - #60                 5 - #65                 5 -   
#70                                                                       
- att gaa cca tta gga gta gca ccc atc taggcaaag - #a gaagagtggt           
2439                                                                      
Ile Glu Pro Leu Gly Val Ala Pro Ile                                       
                575                                                       
- gcagagagaa aaaagagcag tgacactagg agctatgttc cttgggttct tg - #ggagcagc   
2499                                                                      
- aggaagcact atgggcgata agctttaatg cggtagttta tcacagttaa at - #tcgtaacg   
2559                                                                      
- cagtcaggca ccgtgtatga aatctaacaa tgcgacctgc agaagcttag aa - #ccgaggaa   
2619                                                                      
- cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa at - #ttcacaaa   
2679                                                                      
- taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca at - #gtatctta   
2739                                                                      
- tcatgtctgg atcgggaatt aattcggcgc agcaccatgg cctgaaataa cc - #tctgaaag   
2799                                                                      
#             2800                                                        
- <210> SEQ ID NO 43                                                      
<211> LENGTH: 50                                                          
<212> TYPE: PRT                                                           
<213> ORGANISM: HIV                                                       
- <400> SEQUENCE: 43                                                      
- Ser Val Ser Ser Cys Trp Trp Leu Gln Phe Ar - #g Asn Ser Lys Pro Cys     
#                15                                                       
- Ser Glu Tyr Cys Leu Ser His Leu Val Asn Le - #u Arg Glu Asp Trp Gly     
#            30                                                           
- Pro Cys Asp Lys Leu Gln Arg Glu Arg Pro Th - #r Thr Pro Ile Ile Ser     
#        45                                                               
- Tyr Pro                                                                 
    50                                                                    
- <210> SEQ ID NO 44                                                      
<211> LENGTH: 528                                                         
<212> TYPE: PRT                                                           
<213> ORGANISM: HIV                                                       
- <400> SEQUENCE: 44                                                      
- Gly Leu Phe Cys Val Val Arg Ser Gly Met Gl - #y Gly Thr Ala Ala Arg     
#                15                                                       
- Leu Gly Ala Val Ile Leu Phe Val Val Ile Va - #l Gly Leu His Gly Val     
#            30                                                           
- Arg Gly Lys Tyr Ala Leu Ala Asp Ala Ser Le - #u Lys Met Ala Asp Pro     
#        45                                                               
- Asn Arg Phe Arg Gly Lys Asp Leu Pro Val Le - #u Asp Gln Leu Leu Glu     
#    60                                                                   
- Val Pro Val Trp Lys Glu Ala Asn Thr Thr Le - #u Phe Cys Ala Ser Asp     
#80                                                                       
- Ala Lys Ala Tyr Lys Thr Glu Ala His Asn Va - #l Trp Ala Thr His Ala     
#                95                                                       
- Cys Val Pro Thr Asp Pro Lys Pro Gln Glu Il - #e Lys Leu Glu Asn Val     
#           110                                                           
- Thr Glu Asn Phe Asn Met Trp Lys Asn Asn Me - #t Val Glu Gln Met His     
#       125                                                               
- Glu Asp Ile Ile Ser Leu Trp Asp Gln Ser Le - #u Lys Pro Cys Val Lys     
#   140                                                                   
- Leu Thr Pro Leu Cys Val Thr Leu Asn Cys Th - #r Asp Leu Arg Asn Asn     
145                 1 - #50                 1 - #55                 1 -   
#60                                                                       
- Thr Asn Thr Asn Ser Thr Tyr Gly Lys Ile Me - #t Glu Gly Gly Glu Ile     
#               175                                                       
- Lys Asn Cys Ser Phe Asn Ile Thr Thr Ser Il - #e Lys Asp Lys Leu Lys     
#           190                                                           
- Asp Met Ser Leu Phe Tyr Lys Leu Asp Val Va - #l Pro Ile Gly Asn Asn     
#       205                                                               
- Ser Asn Thr Thr Ser Tyr Arg Leu Ile Ser Cy - #s Asn Thr Ser Val Ile     
#   220                                                                   
- Thr Gln Ala Cys Pro Lys Thr Ser Phe Glu Pr - #o Ile Pro Ile His Tyr     
225                 2 - #30                 2 - #35                 2 -   
#40                                                                       
- Cys Ala Pro Ala Gly Phe Ala Ile Leu Lys Cy - #s Asn Asp Asn Lys Phe     
#               255                                                       
- Asn Gly Thr Gly Pro Cys Pro Asn Val Ser Th - #r Val Gln Cys Thr His     
#           270                                                           
- Gly Ile Arg Pro Val Val Ser Thr Gln Leu Le - #u Leu Asn Gly Ser Leu     
#       285                                                               
- Ala Glu Lys Glu Val Val Leu Arg Ser Glu As - #n Phe Thr Asp Asn Ala     
#   300                                                                   
- Lys Thr Ile Ile Val Gln Leu Asn Glu Ser Va - #l Ile Ile Asp Cys Met     
305                 3 - #10                 3 - #15                 3 -   
#20                                                                       
- Arg Pro Asn Asn Asn Thr Arg Thr Ser Ile Pr - #o Met Gly Pro Gly Lys     
#               335                                                       
- Ala Phe Tyr Ala Thr Gly Asp Val Ile Gly As - #p Ile Arg Arg Ala His     
#           350                                                           
- Cys Asn Ile Ser Arg Ala Gly Trp Asn Thr Th - #r Leu Gln Gln Ile Ala     
#       365                                                               
- Lys Lys Leu Arg Glu Lys Phe Glu Asn Lys Th - #r Ile Val Phe Asn His     
#   380                                                                   
- Ser Ser Gly Gly Asp Pro Glu Ile Val Met Hi - #s Thr Phe Asn Cys Gly     
385                 3 - #90                 3 - #95                 4 -   
#00                                                                       
- Gly Glu Phe Phe Cys Cys Asn Ser Thr Pro Le - #u Phe Asn Ser Thr Trp     
#               415                                                       
- Asn Asp Ala Gln Leu Phe Asn Ser Thr Trp As - #p Asp Thr Lys Trp Ser     
#           430                                                           
- Lys Gly Thr Asn Glu Asn Asp Thr Ile Thr Le - #u His Cys Arg Ile Lys     
#       445                                                               
- Gln Ile Ile Asn Met Trp Gln Glu Val Gly Ly - #s Ala Met Tyr Ala Pro     
#   460                                                                   
- Pro Ile Lys Gly Gln Ile Arg Cys Glu Ser As - #n Ile Thr Gly Leu Leu     
465                 4 - #70                 4 - #75                 4 -   
#80                                                                       
- Leu Thr Arg Asp Gly Gly Asn Asp Thr Ser Ly - #s Asn Asn Thr Glu Ile     
#               495                                                       
- Phe Arg Pro Gly Gly Gly Asn Met Lys Asp As - #n Trp Arg Ser Glu Leu     
#           510                                                           
- Tyr Lys Tyr Lys Val Ile Lys Ile Glu Pro Le - #u Gly Val Ala Pro Ile     
#       525                                                               
- <210> SEQ ID NO 45                                                      
<211> LENGTH: 1533                                                        
<212> TYPE: DNA                                                           
<213> ORGANISM: HIV                                                       
<220> FEATURE:                                                            
<221> NAME/KEY: CDS                                                       
<222> LOCATION: (166)...(1533)                                            
- <400> SEQUENCE: 45                                                      
- atggggggga ctgccgccag gttgggggcc gtgattttgt ttgtcgtcat ag - #tgggcctc   
  60                                                                      
- catggggtcc gcggcaaata tgccttggcg gatgcctctc tcaagatggc cg - #accccaat   
 120                                                                      
#cct gtg tgg    177acct tccggtcctg gaccagctgc tcgag gta                   
#              Val Pro V - #al Trp                                        
#               1                                                         
- aaa gaa gca acc acc act cta ttt tgt gca tc - #a gat gct aaa gca tat     
 225                                                                      
Lys Glu Ala Thr Thr Thr Leu Phe Cys Ala Se - #r Asp Ala Lys Ala Tyr       
#  20                                                                     
- gat aca gag gta cat aat gtt tgg gcc aca ca - #t gcc tgt gta ccc aca     
 273                                                                      
Asp Thr Glu Val His Asn Val Trp Ala Thr Hi - #s Ala Cys Val Pro Thr       
#                 35                                                      
- gac ccc aac cca caa gaa ata gga ttg gaa aa - #t gta aca gaa aat ttt     
 321                                                                      
Asp Pro Asn Pro Gln Glu Ile Gly Leu Glu As - #n Val Thr Glu Asn Phe       
#             50                                                          
- aac atg tgg aaa aat aac atg gta gaa cag at - #g cat gag gat ata atc     
 369                                                                      
Asn Met Trp Lys Asn Asn Met Val Glu Gln Me - #t His Glu Asp Ile Ile       
#         65                                                              
- agt tta tgg gat caa agc tta aag cca tgt gt - #a aaa tta acc cca cta     
 417                                                                      
Ser Leu Trp Asp Gln Ser Leu Lys Pro Cys Va - #l Lys Leu Thr Pro Leu       
#     80                                                                  
- tgt gtt act tta aat tgc act gat ttg aaa aa - #t gct act aat acc act     
 465                                                                      
Cys Val Thr Leu Asn Cys Thr Asp Leu Lys As - #n Ala Thr Asn Thr Thr       
#100                                                                      
- agt agc agc tgg gga aag atg gag aga gga ga - #a ata aaa aac tgc tct     
 513                                                                      
Ser Ser Ser Trp Gly Lys Met Glu Arg Gly Gl - #u Ile Lys Asn Cys Ser       
#               115                                                       
- ttc aat gtc acc aca agt ata aga gat aag at - #g aag aat gaa tat gca     
 561                                                                      
Phe Asn Val Thr Thr Ser Ile Arg Asp Lys Me - #t Lys Asn Glu Tyr Ala       
#           130                                                           
- ctt ttt tat aaa ctt gat gta gta cca ata ga - #t aat gat aat act agc     
 609                                                                      
Leu Phe Tyr Lys Leu Asp Val Val Pro Ile As - #p Asn Asp Asn Thr Ser       
#       145                                                               
- tat agg ttg ata agt tgt aac acc tca gtc at - #t aca cag gcc tgt cca     
 657                                                                      
Tyr Arg Leu Ile Ser Cys Asn Thr Ser Val Il - #e Thr Gln Ala Cys Pro       
#   160                                                                   
- aag gtg tcc ttt gag cca att ccc ata cat ta - #t tgt gcc ccg gct ggt     
 705                                                                      
Lys Val Ser Phe Glu Pro Ile Pro Ile His Ty - #r Cys Ala Pro Ala Gly       
165                 1 - #70                 1 - #75                 1 -   
#80                                                                       
- ttt gcg att cta aag tgt aga gat aaa aag tt - #c aac gga aca gga cca     
 753                                                                      
Phe Ala Ile Leu Lys Cys Arg Asp Lys Lys Ph - #e Asn Gly Thr Gly Pro       
#               195                                                       
- tgt aca aat gtc agc aca gta caa tgt aca ca - #t gga att agg cca gta     
 801                                                                      
Cys Thr Asn Val Ser Thr Val Gln Cys Thr Hi - #s Gly Ile Arg Pro Val       
#           210                                                           
- gta tca act caa ctg ctg tta aat ggc agt tt - #a gca gaa gaa gaa gta     
 849                                                                      
Val Ser Thr Gln Leu Leu Leu Asn Gly Ser Le - #u Ala Glu Glu Glu Val       
#       225                                                               
- gta att aga tct gcc aat ttc tcg gac aat gc - #t aaa acc ata ata gta     
 897                                                                      
Val Ile Arg Ser Ala Asn Phe Ser Asp Asn Al - #a Lys Thr Ile Ile Val       
#   240                                                                   
- cag ctg aac gaa tct gta gaa att aat tgt ac - #a aga ccc aac aac aat     
 945                                                                      
Gln Leu Asn Glu Ser Val Glu Ile Asn Cys Th - #r Arg Pro Asn Asn Asn       
245                 2 - #50                 2 - #55                 2 -   
#60                                                                       
- aca aga aga agt ata cat ata gga cca ggg ag - #a gca ttt tat gca aca     
 993                                                                      
Thr Arg Arg Ser Ile His Ile Gly Pro Gly Ar - #g Ala Phe Tyr Ala Thr       
#               275                                                       
- gga gaa ata ata gga gac ata aga caa gca ca - #t tgt aac ctt agt agc     
1041                                                                      
Gly Glu Ile Ile Gly Asp Ile Arg Gln Ala Hi - #s Cys Asn Leu Ser Ser       
#           290                                                           
- aca aaa tgg aat aat act tta aaa cag ata gt - #t aca aaa tta aga gaa     
1089                                                                      
Thr Lys Trp Asn Asn Thr Leu Lys Gln Ile Va - #l Thr Lys Leu Arg Glu       
#       305                                                               
- cat ttt aat aaa aca ata gtc ttt aat cac tc - #c tca gga ggg gac cca     
1137                                                                      
His Phe Asn Lys Thr Ile Val Phe Asn His Se - #r Ser Gly Gly Asp Pro       
#   320                                                                   
- gaa att gta atg cac agt ttt aat tgt gga gg - #g gaa ttt ttc tac tgt     
1185                                                                      
Glu Ile Val Met His Ser Phe Asn Cys Gly Gl - #y Glu Phe Phe Tyr Cys       
325                 3 - #30                 3 - #35                 3 -   
#40                                                                       
- aat aca aca cca ctg ttt aat agt act tgg aa - #t tat act tat act tgg     
1233                                                                      
Asn Thr Thr Pro Leu Phe Asn Ser Thr Trp As - #n Tyr Thr Tyr Thr Trp       
#               355                                                       
- aat aat act gaa ggg tca aat gac act gga ag - #a aat atc aca ctc caa     
1281                                                                      
Asn Asn Thr Glu Gly Ser Asn Asp Thr Gly Ar - #g Asn Ile Thr Leu Gln       
#           370                                                           
- tgc aga ata aaa caa att ata aac atg tgg ca - #g gaa gta gga aaa gca     
1329                                                                      
Cys Arg Ile Lys Gln Ile Ile Asn Met Trp Gl - #n Glu Val Gly Lys Ala       
#       385                                                               
- atg tat gcc cct ccc ata aga gga caa att ag - #a tgc tca tca aat att     
1377                                                                      
Met Tyr Ala Pro Pro Ile Arg Gly Gln Ile Ar - #g Cys Ser Ser Asn Ile       
#   400                                                                   
- aca ggg ctg cta tta aca aga gat ggt ggt aa - #t aac agc gaa acc gag     
1425                                                                      
Thr Gly Leu Leu Leu Thr Arg Asp Gly Gly As - #n Asn Ser Glu Thr Glu       
405                 4 - #10                 4 - #15                 4 -   
#20                                                                       
- atc ttc aga cct gga gga gga gat atg agg ga - #c aat tgg aga agt gaa     
1473                                                                      
Ile Phe Arg Pro Gly Gly Gly Asp Met Arg As - #p Asn Trp Arg Ser Glu       
#               435                                                       
- tta tat aaa tat aaa gta gta aaa att gaa cc - #a tta gga gta gca ccc     
1521                                                                      
Leu Tyr Lys Tyr Lys Val Val Lys Ile Glu Pr - #o Leu Gly Val Ala Pro       
#           450                                                           
#     1533                                                                
Thr Lys Ala                                                               
        455                                                               
- <210> SEQ ID NO 46                                                      
<211> LENGTH: 455                                                         
<212> TYPE: PRT                                                           
<213> ORGANISM: HIV                                                       
- <400> SEQUENCE: 46                                                      
- Val Pro Val Trp Lys Glu Ala Thr Thr Thr Le - #u Phe Cys Ala Ser Asp     
#                15                                                       
- Ala Lys Ala Tyr Asp Thr Glu Val His Asn Va - #l Trp Ala Thr His Ala     
#            30                                                           
- Cys Val Pro Thr Asp Pro Asn Pro Gln Glu Il - #e Gly Leu Glu Asn Val     
#        45                                                               
- Thr Glu Asn Phe Asn Met Trp Lys Asn Asn Me - #t Val Glu Gln Met His     
#    60                                                                   
- Glu Asp Ile Ile Ser Leu Trp Asp Gln Ser Le - #u Lys Pro Cys Val Lys     
#80                                                                       
- Leu Thr Pro Leu Cys Val Thr Leu Asn Cys Th - #r Asp Leu Lys Asn Ala     
#                95                                                       
- Thr Asn Thr Thr Ser Ser Ser Trp Gly Lys Me - #t Glu Arg Gly Glu Ile     
#           110                                                           
- Lys Asn Cys Ser Phe Asn Val Thr Thr Ser Il - #e Arg Asp Lys Met Lys     
#       125                                                               
- Asn Glu Tyr Ala Leu Phe Tyr Lys Leu Asp Va - #l Val Pro Ile Asp Asn     
#   140                                                                   
- Asp Asn Thr Ser Tyr Arg Leu Ile Ser Cys As - #n Thr Ser Val Ile Thr     
145                 1 - #50                 1 - #55                 1 -   
#60                                                                       
- Gln Ala Cys Pro Lys Val Ser Phe Glu Pro Il - #e Pro Ile His Tyr Cys     
#               175                                                       
- Ala Pro Ala Gly Phe Ala Ile Leu Lys Cys Ar - #g Asp Lys Lys Phe Asn     
#           190                                                           
- Gly Thr Gly Pro Cys Thr Asn Val Ser Thr Va - #l Gln Cys Thr His Gly     
#       205                                                               
- Ile Arg Pro Val Val Ser Thr Gln Leu Leu Le - #u Asn Gly Ser Leu Ala     
#   220                                                                   
- Glu Glu Glu Val Val Ile Arg Ser Ala Asn Ph - #e Ser Asp Asn Ala Lys     
225                 2 - #30                 2 - #35                 2 -   
#40                                                                       
- Thr Ile Ile Val Gln Leu Asn Glu Ser Val Gl - #u Ile Asn Cys Thr Arg     
#               255                                                       
- Pro Asn Asn Asn Thr Arg Arg Ser Ile His Il - #e Gly Pro Gly Arg Ala     
#           270                                                           
- Phe Tyr Ala Thr Gly Glu Ile Ile Gly Asp Il - #e Arg Gln Ala His Cys     
#       285                                                               
- Asn Leu Ser Ser Thr Lys Trp Asn Asn Thr Le - #u Lys Gln Ile Val Thr     
#   300                                                                   
- Lys Leu Arg Glu His Phe Asn Lys Thr Ile Va - #l Phe Asn His Ser Ser     
305                 3 - #10                 3 - #15                 3 -   
#20                                                                       
- Gly Gly Asp Pro Glu Ile Val Met His Ser Ph - #e Asn Cys Gly Gly Glu     
#               335                                                       
- Phe Phe Tyr Cys Asn Thr Thr Pro Leu Phe As - #n Ser Thr Trp Asn Tyr     
#           350                                                           
- Thr Tyr Thr Trp Asn Asn Thr Glu Gly Ser As - #n Asp Thr Gly Arg Asn     
#       365                                                               
- Ile Thr Leu Gln Cys Arg Ile Lys Gln Ile Il - #e Asn Met Trp Gln Glu     
#   380                                                                   
- Val Gly Lys Ala Met Tyr Ala Pro Pro Ile Ar - #g Gly Gln Ile Arg Cys     
385                 3 - #90                 3 - #95                 4 -   
#00                                                                       
- Ser Ser Asn Ile Thr Gly Leu Leu Leu Thr Ar - #g Asp Gly Gly Asn Asn     
#               415                                                       
- Ser Glu Thr Glu Ile Phe Arg Pro Gly Gly Gl - #y Asp Met Arg Asp Asn     
#           430                                                           
- Trp Arg Ser Glu Leu Tyr Lys Tyr Lys Val Va - #l Lys Ile Glu Pro Leu     
#       445                                                               
- Gly Val Ala Pro Thr Lys Ala                                             
#   455                                                                   
- <210> SEQ ID NO 47                                                      
<211> LENGTH: 37                                                          
<212> TYPE: DNA                                                           
<213> ORGANISM: Artificial Sequence                                       
<220> FEATURE:                                                            
<223> OTHER INFORMATION: Primer                                           
- <400> SEQUENCE: 47                                                      
#      37          agca gaagacagtg gcaatga                                
- <210> SEQ ID NO 48                                                      
<211> LENGTH: 33                                                          
<212> TYPE: DNA                                                           
<213> ORGANISM: Artificial Sequence                                       
<220> FEATURE:                                                            
<223> OTHER INFORMATION: Primer                                           
- <400> SEQUENCE: 48                                                      
#         33       cagt cagactcatc aag                                    
- <210> SEQ ID NO 49                                                      
<211> LENGTH: 39                                                          
<212> TYPE: DNA                                                           
<213> ORGANISM: Artificial Sequence                                       
<220> FEATURE:                                                            
<223> OTHER INFORMATION: Primer                                           
- <400> SEQUENCE: 49                                                      
#    39            ccca tagtgcttcc tgctgctcc                              
- <210> SEQ ID NO 50                                                      
<211> LENGTH: 36                                                          
<212> TYPE: DNA                                                           
<213> ORGANISM: Artificial Sequence                                       
<220> FEATURE:                                                            
<223> OTHER INFORMATION: Primer                                           
- <400> SEQUENCE: 50                                                      
#       36         tacc tgtrtggaaa gaagca                                 
- <210> SEQ ID NO 51                                                      
<211> LENGTH: 38                                                          
<212> TYPE: DNA                                                           
<213> ORGANISM: Artificial Sequence                                       
<220> FEATURE:                                                            
<223> OTHER INFORMATION: Primer                                           
- <400> SEQUENCE: 51                                                      
#     38           gctc cyaagaaccc aaggaaca                               
- <210> SEQ ID NO 52                                                      
<211> LENGTH: 7                                                           
<212> TYPE: PRT                                                           
<213> ORGANISM: HIV                                                       
- <400> SEQUENCE: 52                                                      
- Ile Gly Pro Gly Arg Ala Phe                                             
 1               5                                                        
- <210> SEQ ID NO 53                                                      
<211> LENGTH: 7                                                           
<212> TYPE: PRT                                                           
<213> ORGANISM: HIV                                                       
- <400> SEQUENCE: 53                                                      
- Ile Gly Pro Gly Arg Ala Trp                                             
 1               5                                                        
- <210> SEQ ID NO 54                                                      
<211> LENGTH: 7                                                           
<212> TYPE: PRT                                                           
<213> ORGANISM: HIV                                                       
- <400> SEQUENCE: 54                                                      
- Leu Gly Pro Gly Ser Thr Phe                                             
 1               5                                                        
- <210> SEQ ID NO 55                                                      
<211> LENGTH: 7                                                           
<212> TYPE: PRT                                                           
<213> ORGANISM: HIV                                                       
- <400> SEQUENCE: 55                                                      
- Ile Gly Pro Gly Arg Val Leu                                             
 1               5                                                        
- <210> SEQ ID NO 56                                                      
<211> LENGTH: 7                                                           
<212> TYPE: PRT                                                           
<213> ORGANISM: HIV                                                       
- <400> SEQUENCE: 56                                                      
- Ile Gly Pro Gly Ser Ala Phe                                             
 1               5                                                        
- <210> SEQ ID NO 57                                                      
<211> LENGTH: 5                                                           
<212> TYPE: PRT                                                           
<213> ORGANISM: HIV                                                       
- <400> SEQUENCE: 57                                                      
- Ile Gly Pro Gly Arg                                                     
 1               5                                                        
__________________________________________________________________________

Claims (33)

What is claimed is:
1. An isolated polypeptide comprising an HIV gp 120 amino acid sequence selected from the group consisting of Sequence ID Nos: 2, 5, 8, 10, 12, 16, 19, 23, 25, 28, 31, 33, 36, and 39, and fragments thereof, wherein each of said fragments comprises at least the V2, V3, and C4 domains of gp120.
2. The polypeptide of claim 1 wherein the polypeptide additionally comprises a flag epitope sequence.
3. The polypeptide of claim 2 wherein the flag epitope sequence is the HSV gD-1 flag epitope sequence.
4. The polypeptide of claim 2 wherein the flag epitope sequence is fused to the HIV gp120 amino acid sequence.
5. The polypeptide of claim 1 wherein said HIV gp 120 amino acid sequence is a fragment lacking the gp120 signal sequence.
6. The polypeptide of claim 5 wherein the polypeptide additionally comprises a flag epitope sequence.
7. The polypeptide of claim 5 wherein the flag epitope sequence is the HSV gD-1 flag epitope sequence.
8. The polypeptide of claim 1 wherein the polypeptide comprises an amino acid sequence selected from the group consisting of Sequence ID Nos. 2 and 5 and fragments thereof, wherein each of said fragments comprises at least the V2, V3, and C4 domains of gp120.
9. The polypeptide of claim 1 wherein the polypeptide comprises an amino acid sequence selected from the group consisting of Sequence ID Nos. 8 and 10 and fragments thereof, wherein each of said fragments comprises at least the V2, V3, and C4 domains of gp120.
10. The polypeptide of claim 1 wherein the polypeptide comprises an amino acid sequence selected from the group consisting of Sequence ID Nos. 12 and 16 and fragments thereof, wherein each of said fragments comprises at least the V2, V3, and C4 domains of gp120.
11. The polypeptide of claim 1 wherein the polypeptide comprises an amino acid sequence selected from the group consisting of Sequence ID Nos. 19 and 23 and fragments thereof, wherein each of said fragments comprises at least the V2, V3, and C4 domains of gp120.
12. The polypeptide of claim 1 wherein the polypeptide comprises an amino acid sequence selected from the group consisting of Sequence ID Nos. 25 and 28 and fragments thereof, wherein each of said fragments comprises at least the V2, V3, and C4 domains of gp120.
13. The polypeptide of claim 1 wherein the polypeptide comprises an amino acid sequence selected from the group consisting of Sequence ID Nos. 31 and 33 and fragments thereof, wherein each of said fragments comprises at least the V2, V3, and C4 domains of gp120.
14. The polypeptide of claim 1 wherein the polypeptide comprises an amino acid sequence selected from the group consisting of Sequence ID Nos. 36 and 39 and fragments thereof, wherein each of said fragments comprises at least the V2, V3, and C4 domains of gp120.
15. An oligonucleotide of not more than five kilobases encoding an HIV gp120 polypeptide sequence comprising an amino acid sequence selected from the group consisting of Sequence ID Nos. 2, 5, 8, 10, 12, 16, 19, 23, 25, 28, 31, 33, 36, and 39, and fragments thereof, wherein each of said fragments encodes at least the V2, V3, and C4 domains of gp120.
16. The oligonucleotide of claim 15 wherein the oligonucleotide additionally encodes a flag epitope.
17. The oligonucleotide of claim 15 wherein the flag epitope is the HSV gD-1 flag epitope.
18. The oligonucleotide of claim 16 wherein the sequence encoding the flag epitope is fused to the sequence encoding the HIV gp120 amino acid sequence.
19. The oligonucleotide of claim 15 wherein said amino acid sequence is a fragment lacking the gp120 signal sequence.
20. The oligonucleotide of claim 19 wherein the nucleotide sequence encoding said fragment is joined to a nucleotide sequence encoding a heterologous signal sequence.
21. The oligonucleotide of claim 20 wherein said nucleotide sequences are joined via a nucleotide sequence encoding a flag epitope sequence.
22. The oligonucleotide of claim 20 wherein the signal sequence is the HSV gD1 signal sequence and the flag epitope sequence is the HSV gD-1 flag epitope sequence.
23. A vector comprising the oligonucleotide of claim 15.
24. A host cell comprising the vector of claim 23.
25. A method of producing a polypeptide comprising culturing the host cell of claim 24 and recovering the polypeptide.
26. An oligonucleotide of not more than 5 kilobases comprising a nucleotide sequence selected from the group consisting of Sequence ID Nos: 1, 4, 7, 9, 11, 15, 18, 22, 24, 27, 30, 32, 35, and 38, and fragments thereof, wherein each of said fragments encodes at least the V2, V3, and C4 domains of gp120.
27. The oligonucleotide of claim 26 wherein the oligonucleotide comprises a nucleotide sequence selected from the group consisting of Sequence ID Nos. 1 and 4 and fragments thereof, wherein each of said fragments encodes at least the V2, V3, and C4 domains of gp120.
28. The oligonucleotide of claim 26 wherein the oligonucleotide comprises a nucleotide sequence selected from the group consisting of Sequence ID Nos. 7 and 9 and fragments thereof, wherein each of said fragments encodes at least the V2, V3, and C4 domains of gp120.
29. The oligonucleotide of claim 26 wherein the oligonucleotide comprises a nucleotide sequence selected from the group consisting of Sequence ID Nos. 11 and 15 and fragments thereof, wherein each of said fragments encodes at least the V2, V3, and C4 domains of gp120.
30. The oligonucleotide of claim 26 wherein the oligonucleotide comprises a nucleotide sequence selected from the group consisting of Sequence ID Nos. 18 and 22 and fragments thereof, wherein each of said fragments encodes at least the V2, V3, and C4 domains of gp120.
31. The oligonucleotide of claim 26 wherein the oligonucleotide comprises a nucleotide sequence selected from the group consisting of Sequence ID Nos. 24 and 27 and fragments thereof, wherein each of said fragments encodes at least the V2, V3, and C4 domains of gp120.
32. The oligonucleotide of claim 26 wherein the oligonucleotide comprises a nucleotide sequence selected from the group consisting of Sequence ID Nos. 30 and 32 and fragments thereof, wherein each of said fragments encodes at least the V2, V3, and C4 domains of gp120.
33. The oligonucleotide of claim 26 wherein the oligonucleotide comprises a nucleotide sequence selected from the group consisting of Sequence ID Nos. 35 and 38 and fragments thereof, wherein each of said fragments encodes at least the V2, V3, and C4 domains of gp120.
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US10/371,472 US7071322B2 (en) 1996-07-08 2003-02-21 HIV envelope polynucleotides and immunogenic composition

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